“Ziziphus oxyphylla”: Ethnobotanical, ethnopharmacological and phytochemical review

“Ziziphus oxyphylla”: Ethnobotanical, ethnopharmacological and phytochemical review

Biomedicine & Pharmacotherapy 91 (2017) 970–998 Available online at ScienceDirect www.sciencedirect.com “Ziziphus oxyphylla”: Ethnobotanical, ethno...

2MB Sizes 0 Downloads 24 Views

Biomedicine & Pharmacotherapy 91 (2017) 970–998

Available online at

ScienceDirect www.sciencedirect.com

“Ziziphus oxyphylla”: Ethnobotanical, ethnopharmacological and phytochemical review Rizwan Ahmada,* , Niyaz Ahmadb , Atta Abbas Naqvic a b c

Natural Products and Alternative Medicine, College of Clinical Pharmacy, Imam Abdulrahman Bin Faisal University, Saudi Arabia Department of Pharmaceutics, College of Clinical Pharmacy, Imam Abdulrahman Bin Faisal University, Saudi Arabia Department of Pharmacy Practice, College of Clinical Pharmacy, Imam Abdulrahman Bin Faisal University, Saudi Arabia

A R T I C L E I N F O

A B S T R A C T

Article history: Received 24 March 2017 Received in revised form 26 April 2017 Accepted 30 April 2017

Ethnopharmacological relevance: Ziziphus oxyphylla (ZO) is distributed mainly in tropic and warm temperate regions in the world. Pakistan owns six (06) indigenous species of genus Ziziphus out of which ZO is widely used for traditional treatment of different ailments such as diabetes, jaundice and liver diseases. Aim of the study: The present review aims to provide in-depth and comprehensive literature overview, regarding botanical, chemical and biological characteristics of the plant alongwith phytochemical isolation and mechanistic studies to support its folklore and traditional uses. Materials and methods: The literature search and relevant information were collected through authentic resources using data bases such as Google Scholar, PubMed, Web of Science, Scopus and Science Direct, peer reviewed articles, books and thesis. Results and discussion: The phytochemical characterization as well as color tests confirmed the presence of diverse chemical groups presents in the plant such as alkaloids, flavonoids, phenolic compounds and tannins. In-vivo and in-vitro pharmacological activities for the crude extracts and its fractions revealed potent antinociceptive, anti-inflammatory, antipyretic, antioxidant, antibacterial as well as acetyl choline esterase and lipoxygenase inhibitory activity. Majority of the isolated compounds belonged to class of Cyclopeptide alkaloids for which the genus is already very famous. Compounds from alkaloids and flavonoids chemical class were isolated and evaluated with a role as antioxidant, antidiabetic, antiglycation and advanced glycation end products inhibitors. No toxicity was observed during cytotoxicity (MRC-5 cell lines), insecticidal and brine shrimp lethality studies. Conclusion: The review article supports the folklore uses of this plant in the aforementioned diseases. The plant due to its diverse biological nature may be further studied for mechanistic studies, its anticancer effects as well as its potency and toxicity studies for safe use in human beings. © 2017 Elsevier Masson SAS. All rights reserved.

Keywords: Ethnobotanical Ethnopharmacological Cyclopeptide alkaloids Flavonoids Ziziphus oxyphylla Rhamnaceae

Contents 1. 2. 3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Materials and methods . . . . . . . . . . . . . . . . . . . . . Literature review . . . . . . . . . . . . . . . . . . . . . . . . . . Plant description . . . . . . . . . . . . . . . . . . . . 3.1. Plant . . . . . . . . . . . . . . . . . . . . . 3.1.1. Leaves . . . . . . . . . . . . . . . . . . . . 3.1.2. 3.1.3. Flower . . . . . . . . . . . . . . . . . . . Calyx . . . . . . . . . . . . . . . . . . . . . 3.1.4. Fruit . . . . . . . . . . . . . . . . . . . . . 3.1.5. General pollen characters for Z. oxyphylla 3.2.

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

* Corresponding author. E-mail addresses: [email protected], [email protected] (R. Ahmad). http://dx.doi.org/10.1016/j.biopha.2017.04.129 0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

971 971 972 972 972 972 972 972 972 973

R. Ahmad et al. / Biomedicine & Pharmacotherapy 91 (2017) 970–998

3.3. 3.4. 3.5. 3.6. 3.7.

4.

Geographical distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Vernacular names . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Part used . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Elemental analysis of Ziziphus oxyphylla . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Phytochemical screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Color reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7.1. Quantitative tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7.2. Phytochemical tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7.3. 3.7.4. Total phenol content, flavonoid content and antioxidant activity (TPC, TFC, TAA) Mode of preparation and application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.8. Ethnobotanical uses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.9. 3.10. Ethnomedicinal uses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Phytochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.11. Identification of phenolic compounds via HPLC-ESI–MS/MS analysis . . . . . . . . . 3.11.1. Cyclopeptide alkaloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.11.2. 3.11.3. Flavonoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structures of bioassay-guided isolated compounds from Ziziphus oxyphylla . . . . . . . . . . . . . 3.12. Pharmacological activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.13. Antioxidant activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.13.1. 3.13.2. Analgesic and antinociceptive activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Antipyretic activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.13.3. Anti-inflammatory activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.13.4. Antiglycation activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.13.5. 3.13.6. Antidiabetic property . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Antibacterial activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.13.7. Anti-fungal activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.13.8. Toxicity studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.13.9. Phytotoxicity studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.13.10. Antileishmanial activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.13.11. Antiplasmodial activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.13.12. Antitrypanosomal activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.13.13. Acetyl choline esterase inhibitory activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.13.14. Lipoxygenase (LOX) inhibitory activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.13.15. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ethics approval and consent to participate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Consent for publication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Authors contribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Funding source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction The recent literature reports as well as advancement in the field of alternative treatment systems is a self-supportive evidence for increased use of herbs. Plants in the form of natural products or dietary supplements have been prescribed for the treatment of various ailments, since ages. This high versatility imparts to plants the characteristics of being utilized in different traditional systems [1]. The traditional herbal treatment system consists of knowledge as well as practices based on such beliefs which may vary among natives of different communities [2]. The genus Ziziphus consists of almost 100 species been used in folk and alternative systems of treatment in order to combat different diseases such as; fever, diabetes, skin infections [3–5] antipyretic, antinociceptive [6,7] antioxidant, antilisterial [8] and larvicidal [9]. Due to peculiar geographical region, Pakistan exhibits a great diversity of flora as represented by 7 genera alongwith 13 species for Ziziphus [10]. Ziziphus oxyphylla (ZO) Edgew (synonym; Ziziphus acuminata Royle) also written as “Zizyphus oxyphylla Edgew” belongs to the genus Ziziphus and family Rhamnaceae (known as buckthorn family). The plant ZO, popular with common names i.e. Mamyanu, Elanai, Bera, Tukbari, Phitni, Amlai and Sezen is a small glabrous tree with short, recurved and unequal spine alongwith edible fruit (oval in shape), belongs to the genus Ziziphus. Based on folklore use,

971

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

973 973 973 973 973 974 974 974 974 974 975 976 977 977 977 979 979 982 982 982 994 994 994 994 994 995 995 996 996 996 996 996 996 996 996 997 997 997 997 997 997

the plant is in-use for traditional treatment of diseases i.e. jaundice [11], diabetes [12], hypertension [13] as well as in gas troubles [14] since long. These traditional and folk uses are transferred ancestrally hence proper ethnobotanical and ethnopharmacological studies are worth to expose and preserve the knowledge regarding the use of such traditional plants in ancient times as well as its current usage in different population of the world. Although a progressive research work and literature report is on the way for ZO, till date no review article with proper information regarding the reported ethnobotanical, ethnobiological and phytochemical studies as well as folklore uses is available. In order to accomplish the mentioned goal, current review article aims to explore different aspects i.e. ethnobotanical, ethnopharmacological and phytochemical, of ZO with regard to its folklore use alongwith current research work carried out on the plant so far. 2. Materials and methods The databases used for literature search include; Google Scholar, Scopus, Web of Science, Science Direct, Springer Link, Sci Finder and PubMed. The terminologies used in the review articles consists of Keywords such as “Ethnomedicinal”; “Ethnobotanical”; “Ethopharmacological”; “Phytochemical”; “Pharmacological”; “Antibacterial”; “Antifungal”. “Anti-infective”; “Antioxidant”; “Cytotoxicity”; “Flavonoids” and “Alkaloids”. The

972

R. Ahmad et al. / Biomedicine & Pharmacotherapy 91 (2017) 970–998

review reports every aspect of plant including folk uses; surveys; ethnobotanical; ethnomedicinal and ethnopharmacological uses. The literature searched is categorized under headings with detail explanation under individual section as well as respective tables for summarization of data as follows;

3. Literature review 3.1. Plant description The plant “Ziziphus oxyphylla”, is a large shrub to medium sized tree [15]. The whole plant as well as leaves and fruits for the plants are shown in Fig. 1A, B and C respectively. The taxonomy i.e. Table 1 and brief plant description for Z. oxyphylla is as below; 3.1.1. Plant It is a small glabrous shrub with main identification of the presence of stipular, recurved and unequal size spines. Some of the spines are too smaller whereas others are slightly recurved with a length of 1.5 mm [16–18]. 3.1.2. Leaves The plants carries leaves with a cordate base and length that ranges normally from 2.5 to 6 cm  1.5 to 3 cm. Leaves also have a cordate, acuminate, glabrous, ovate to lanceolate and serrate to crenately serrate morphology. The petiole has a length of 5–10 mm [16–18].

Table 1 Taxonomy of Ziziphus oxyphylla. Ziziphus oxyphylla Kingdom Subkingdom Superdivision Division Class Subclass Order Family Genus Species Botanical name

Plantae Tracheobionta Spermatophyta Magnoliophyta Magnoliopsida Rosidae Rhamnales Rhamnaceae Ziziphus Oxyphylla Ziziphus oxyphylla Edgew

3.1.3. Flower The cymes of plant are glabrous, fascicled, as well as manyflowered with a diameter of 3 to 4 mm in diameter. The pedicel is wiry, 1 to 2 mm lengthy and glabrous [16–18]. 3.1.4. Calyx The calyx of plant is always 5-lobed, glabrous, without keeling and with a length of 2 mm however the calyx is obtuse to ovate or subacute mostly. The petals carry a length of 1.5 mm which are hooded and spathulate [16–18]. 3.1.5. Fruit The fruit is very fleshy with ovoid (8–10 mm length) nature. The immature fruits may be found as green whereas the ripe fruit as red

Fig. 1. A. Leaves and fruits. B. Whole plant of Ziziphus oxyphylla. C. The whole plant representing different parts of the plants [16].

R. Ahmad et al. / Biomedicine & Pharmacotherapy 91 (2017) 970–998

or orange-black in color. Fruit are with flattened pyrene and are 1 celled and 1 seeded [19,17,18]. 3.2. General pollen characters for Z. oxyphylla The morphology of pollen grain for Rhamnaceae family is quite helpful at generic as well as specific level. The pollen morphology of family Rhamnaceae and genus Ziziphus, as seen through scanning electron microscopy revealed a different morphology. Perveen and Qaiser reported a striate pollen for ZO with secondary characteristics as presented in Table 2 and Fig. 2. These taxa are delimited easily on the basis of exine thickness and polar length [10]. 3.3. Geographical distribution This specie is widely distributed in the warm temperate and subtropical regions throughout the world, especially in Pakistan and India. In Pakistan it may be found in different regions, particularly the rainy and mountain areas as well as the Himalayan series of mountains. The plant is distributed as; Swat Valley, Northern Pakistan [20], Chagharzai valley, District Buner, Khyber Pukhtoon khwa [14], Buner, Hazara, Swat, Garhi Habibullah [21], Dir Kohistan valleys, Pakistan [11], Kot Malakand agency [12], Margallah Hills, Islamabad, Pakistan [11], Islamabad [22], subtropical hills of Darazinda, Takht-e-Suleman range Dera Ismail Khan, Pakistan [23], Khyber Pukhtoon khwa [11], Palas Valley, Pakistan [24], Palas valley [25], Kotli, Azad Jammu Kashmir, Pakistan [26], Bana Valley, Kotli, Azad Jammu and Kashmir, Pakistan [27], Poonch Valley Azad Kashmir, Pakistan [28], Azad Kashmir [13] whereas in India the plant is distributed geographically as; Murad pur, Rajouri district, Jammu and Kashmir, India [29], Kishtwar High Altitude National Park in Northwest, Himalaya, Jammu and Kashmir, India [30].

973

3.4. Vernacular names Ziziphus oxyphylla is known with different common names and these synonym vary from one area or district to the other as; “Mamyanu” in Azad Jammu and Kashmir, Pakistan [31], “Elanai” in Dir, Kohistan valley, Khyber Pukhtoon Khwa, Pakistan [11], “Elanai” in Chagaharzai valley, District Buner, Pakistan [14], “Bera” in Kot Malakand Agency, Pakistan [12], “Tukbari” in Poonch valley, Azad Jammu and Kashmir [13], “Phitni” and “Amlai” in Islamabad, Pakistan [22], “Tuckbari” in Azad Jammu and Kashmir [13] as well as “Sezen” in Palas valley, Pakistan [24] as shown in Table 3. 3.5. Part used All the plant parts are used as in traditional treatment system. The most commonly used part are; Roots and fruits [11,12], Fruits and Bark [13], Fruit [22], Root bark [13], leaves and fruit [14] alongwith Fruits, and roots [24] as shown in Table 3. 3.6. Elemental analysis of Ziziphus oxyphylla The nature and amount of elements present in plants, as estimated with the help of atomic absorption spectroscopy, imparts medicinal value to plants in terms of disease treatment. The more diverse the metal are present, makes the importance of plant materials used in herbal traditional treatment system as well as pharmaceutical companies. Niamat et al. reported the atomic absorption spectroscopy results for different parts of ZO as presented in Table 4. The metals i.e. Na, K, Ca and Mg were found in more abundance in leaves, bark and fruit parts of ZO [39]. 3.7. Phytochemical screening The chemical profiling as well as nature of the chemical class present, have a great influence on the medicinal property of the

Fig. 2. Scanning micrographs, A. Polar view, B. Equatorial view, C. Exine pattern. Scale bar = A & B = 10; C = 1 mm.

Table 2 Pollen characteristics of Ziziphus oxyphylla plant. Pollen Characters Pollen class P/E ratio Shape Aperture Exine Ornamentation Outline

Measurements Tricolporate Sub-transverse or semi- erect Oblate-spheroidal or sub-prolate Long elliptic, acute ends Sexine thicker than nexine or thick as nexine Tectum striate More or less circular

Polar Axis (P) Equatorial diameter (E) Exine Sexine Colpi

15 (30.7  1.0) 45.5 mm long 10 (22.5  1.25) 35 mm 3- (4.0) 5 mm thick Thicker than nexine 12 (22.7  0.31) 32.5 mm long

974

R. Ahmad et al. / Biomedicine & Pharmacotherapy 91 (2017) 970–998

Table 3 Vernacular names, part used and edible part of Ziziphus oxyphylla. Common/local names/synonym

Parts used and general uses

Ziziphus

As spiny plant for fencing and hedge. Fruit is edible Medicinal plant used as fodder and for fuel purposes. Also famous as Honey bee plants. It’s a fruit producing plant. The plant is used as fodder, as wood for fuel, as fruit yielding plant, used as timber, utilized for making furniture and agriculture appliances as well as used as a plant for fence purposes. Leaves are browsed by goats. Spiny branches are used in fencing the fields. Fruit Fruit and root Root bark Shrub and roots

Bera Sezen Tukbari or tuckbari Sezen Phitni, Amlai

Edible or nonedible part

Wood, leaves, root, fruit. Wood used as fodder, fuel wood. Shrubs and roots. Fruit, Root, Bark Leaves Leaves and roots. Wood is used as fuel.

Elanai

Bir Cocon beri Mamyanu

References [32] [35] [23] [27] [12] [11] [28] [33] [22] [34] [36] [14] [37] [20] [30] [38] [31]

Table 4 Elemental analysis of Ziziphus oxyphylla leaves, bark and fruit. Species

Na

K

Zn

Fe

Cr

Co

Cu

Ni

Pb

Mn

Ca

Mg

Cd

Z.O leaves Z.O bark Z.O fruits

44.40 54.14 24.50

60.51 57.15 60.90

0.17 0.14 0.11

8.73 8.55 6.71

0.19 0.14 0.17

0.00 0.00 0.04

0.004 0.005 0

0.005 0.003 0.003

0.001 0.003 0.0020

0.1203 0.455 0.0466

41.36 33.17 18.57

32.60 19.78 18.38

0.004 0.003 0.001

plant. Phytochemical studies are very important in order to explore the characteristics of the chemicals present in plant species. Various procedures are used to find the phytochemical profile of the plants as; 3.7.1. Color reaction The methanol crude extract from roots and leaves of ZO was tested for chemical moieties present [40]. Various tests were performed and the results observed showed the presence of important chemical classes i.e. amine, amino acids, alkaloids, phenolic compounds, tannins etc. as shown in Table 5A and B. 3.7.2. Quantitative tests The quantitative tests i.e. saponification value, ester value and iodine value are used to find out the amount of lipid, volatile oil contents as well as the unsaturation present in the crude extracts. The methanol crude extract of ZO leaves and roots were subjected to various quantitative tests [40]. The result revealed the presence of free acids and esters in ZO plant. The crude extract from roots exhibited saponification value (336.6) in the range limit (1 g crude extract = 300–400 of saponification value) as recommended by European Pharmacopoeia, 2012. The leaves crude extract showed a higher saponification value however, both the leaves and roots crude extract shows no peroxide value hence indicates the absence of peroxide radical as shown in Table 6. 3.7.3. Phytochemical tests The phytochemical tests i.e. dragendorff, millons, Wagner, molisch, vanillin reagent etc. are performed to explore the nature of secondary metabolite present in a plant. Various parts of ZO such as fruits, stem, leaves alongwith its fractions (different polarity solvents) were evaluated for phytochemical test [6,41]. Every part of the plant revealed the presence of important secondary metabolites i.e. alkaloids, saponins, tannins and flavonoids however the most significant fractions observed was ethyl acetate

and n-butanol fraction of the methanol crude extract where almost every chemical class of secondary metabolites was observed. The results observed (Table 7) shows the presence of polar compounds which support the traditional uses of such plant with water during the treatment of different ailments, as aforementioned. 3.7.4. Total phenol content, flavonoid content and antioxidant activity (TPC, TFC, TAA) TPC, TFC and TAA are determined in order to express the phenolic and flavonoid nature of the plants responsible for the antioxidant and hence antidiabetic as well as liver protective effects, for which the plant is been used in traditional systems. The leaves and roots as wells as different polarity fractions of the mentioned parts of the plants were studied [42,43,41] and the results expressed as in Table 8. The TPC value as determined for leaves and root crude extract showed a high amount of phenolic compounds in root as compared to leaves crude extract. The amount of TPC present imparts antioxidant activity to this plant. In addition, the crude extracts were also tested for the amount of flavonoid’s present and the result observed for TFC was more in leaves as compared to root crude extract. The results support the free radical scavenging activity of the plant due to presence of high amount of flavonoids present. The TAC too, evaluated as TEAC (Trolox equivalent) and FRAP (Ferric reducing power), revealed significant results particularly for the leaves, fruits and stem crude extracts. 3.8. Mode of preparation and application The mode of preparation of the plant and its usage in specific form reflects the traditional methods of treatment adopted by different communities. Various parts of the plant and different methods of administration have been reported as presented in Table 9. The most widely observed way of preparation is decoction

R. Ahmad et al. / Biomedicine & Pharmacotherapy 91 (2017) 970–998

975

Table 5 A: Color reaction observed for Ziziphus oxyphylla leaves and root crude extracts. B: Color reaction observed for Ziziphus oxyphylla leaves and root crude extracts. A S. No

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Test

Color Observed

CuSO4 test Diazotization FeSO4 (A) FeSO4 (B) Forrest reagent FPN reagent Ferric chloride Froehde reagent Methanolic KOH Mercurous nitrate DDR Liebermann's reagent McNally test Formaldehyde-H2SO4 Marquis test Millon's reagent Nitrous acid Ninhydrin Potassium dichromate Sodium nitroprusside Nessler's reagent Phosphorus test

Phytochemical present

ZORa

ZOLa

Brown (+) Dark brown () Red brown (+) Red () Brown (+) Brown () Yellowish green (+) Dark red brown (+) Brown (+) Yellowish brown () Orange (+) Orange brown (+) Yellowish brown (+) Orange brown (+) Brown (+) Yellowish brown () Orange (+) Brown (+) Green brown (+) Chocolate brown () Brown orange (+) Bright yellow (+)

Yellowish Green (+) Green () Green () Green () Brown (+) Brown () Green (+) Yellowish green (+) Green yellow () Green () Brown Orange (+) Brown (+) Yellowish Green () Yellowish Green (+) Yellowish brown (+) Yellowish brown () Yellow (+) Yellowish Green (+) Yellow brown (+) Yellowish Green () Green () Yellow (+)

Sulfonamides Aromatic amines Nitrates, Nitrites Cyanide Phenothiazines Phenothiazines and related compounds Salicylates and Phenols Opioids and Amphetamines Quinines, Diones and phenol Derivatives Barbiturates like compounds Alkaloids Phenoxyphenypropionic acid derivatives Aminosalicylic acid and derivatives Benzodiazepines Amphetamines Phenols Sulfonamides Amines and Amino acid groups Phenolic compounds and derivatives Ketones and acetaldehyde Amines and Thioamides Presence of Phosphorus

B S. No

Test

Color Observed

Phytochemical present ZORa

ZOLa

23

Amalic acid

Initial color After Evaporation

Orange Black brown ()

Orange Black brown ()

Xanthines

24

Ammonical-Ag Nitrate

At Room temp At 100C

Orange brown Black (+)

Green Greenish brown ()

Carbidopa, Levodopa, Dopamine

25

Aromaticity

Color with Acid Color with NaOH

Red Brown (+)

Yellow Brown (+)

Aromatic ring

26

Benedict's reagent

Initial color After heating

Leave green Brown (+)

Yellow green Yellowish green ()

Sugars

27

Chromotropic acid

Initial color After

Brown No color ()

Dark Green No color ()

Formaldehyde

28

Napthol-Sulfuric acid

Initial (Heat) After dilution

Black Red brown (+)

Sea green Yellowish brown (+)

Steroidal structures

29

Vanillin Reagent

Before heating After heating

Brown Violet (+)

Brown Violet (+)

Barbiturates

a

ZOR & ZOL (Ziziphus oxyphylla roots and leaves).

and particularly in water. The supportive fact for using the plant aqueous decoction in different traditional system of treatment is due to the presence of more polar compounds as observed in different fraction i.e. ethyl acetate and n-butanol, as discussed in the section on phytochemical screening.

3.9. Ethnobotanical uses The term “ethnobotanical uses” refers to the uses of plants for the purposes other than medicinal i.e. general. Various parts of the plants are famous for non-medicinal uses, out of which fruit (due

976

R. Ahmad et al. / Biomedicine & Pharmacotherapy 91 (2017) 970–998

Table 6 Quantitative tests results for Ziziphus oxyphylla root and leaves crude extract. Crude extract

Acid value

Saponification value

Ester value

Peroxide value

ZORa ZOLa

100.98 106.59

336.6 490.875

235.62 384.285

No Results No Results

a

ZOR & ZOL (Ziziphus oxyphylla roots and leaves).

Table 7 Preliminary tests for phytochemical analysis of Ziziphus oxyphylla crude extract and fractions. Plant part or extract used

Phytochemical present/reported

M.Ea n-HEX CHLO E.A n-BUT A.F a

Reference

Alkaloid

Terpenoid

Saponin

Tannin

Phenolic

Flavonoid

Cardiac glycoside

Sugar

Resins

+ + + + + +

 + + + + +

+  + + + 

+   + + 

+ + + + + 

+  + + + 

+   + + 

 +  + + +

+     

[6] [41]

M.E = Methanol extract, n-HEX = n-Hexane, CHLO = Chloroform, E.A = Ethyl acetate, n-But = n-Butanol, A.F = Aqueous Fraction.

Table 8 TAC, TPC, TFC and FRAP results for Ziziphus oxyphylla crude extracts and its fractions. Sample/extract used

TAC

TPC

TFC

Source

[43]

TAA/TEAC

FRAP

TRAP

42.56 mmol of Trolox/g 41.34 mmol of Trolox/g 48.58 mmol of Trolox/g

207.54 mmol of Fe2+/g 254.89 mmol of Fe2+/g 233.00 mmol of Fe2+/g

112.23 mmol of Trolox/g 102.83 mmol of Trolox/g 117.37 mmol of Trolox/g

27.61 mg/g



15.77 mg/g



17.49 mg/g



CME n-HF CF EAF n-BF AF

1.223 mg/ml 0.187 mg/ml 1.723 mg/ml 1.406 mg/ml 1.523 mg/ml 1.138 mg/ml

150.53 mM/ml 21.5 mM/ml 339.5 mM/ml 296.01 mM/ml 125 mM/ml 107.3 mM/ml

– – – – – –

86.67 mg/g 14.3 mg/g 142.65 mg/g 55.06 mg/g 76.19 mg/g 25.33 mg/g

– – – – – –

[41]

ZO roots







7.95

[42]

ZO leaves







0.17 mg GAE/g 0.09 mg GAE/g

ZO fruit ZO leaves ZO stems

mg quercetin equivalent/g 37.41

mg quercetin equivalent/g

*CME = Crude Methanol extract, n-HF = n-Hexane fraction, CF = Chloroform fraction, EAF = Ethyl acetate fraction, n-BF = n-Butanol fraction, AF = Aqueous Fraction.

Table 9 Parts of Ziziphus oxyphylla and its form used for different indications. Part used

Mode of preparation/application

References

Root bark

Aqueous extract of root bark is mixed with extract of Citrus medica (lemon) in equal quantity and given twice a day to cure hypertension. Roots are boiled in water to get decoction which is used against scabies, pustules and diabetes. Grinded roots are also used against jaundice. Powder and Decoction is used in treatment of various ailments i.e. hypertension, bleeding gums, pimples and dandruff. Decoction of fresh root is used orally in order to treat intestinal worms. Decoction is used. 1 kg of roots alongwith some young shoots of Tecoma capensis (Thunb.) Lindlare boiled in 12 l water until half of the water is evaporated. The decoction is stored and taken 2 cups per days to treat hepatitis. Decoction prepared from roots is taken orally to treat pimples and bleeding gums. Bath is performed in water soaked with chopped leaves once in a week time. It is also considered as sacred.

[28]

Roots Fruit, root and bark Roots Roots Roots Leaves

to fleshy and nontoxic nature) is more used by different age peoples [22,24]. Due to spines and thorny nature, the whole plant is very useful as fence or hedge to protect the fields [14]. In addition, the plant is also used as fodder and fuel wood as well as timber for making furniture as mentioned in Table 10.

[27] [20] [34] [37] [36] [38]

3.10. Ethnomedicinal uses The ethnomedicinal refers to “medicinal uses only” of the plants in various traditional systems of treatments. The worth and importance of medicinal herb is based on the available

R. Ahmad et al. / Biomedicine & Pharmacotherapy 91 (2017) 970–998

977

Table 10 Ethnobotanical uses of different parts of Ziziphus oxyphylla. Parts used and general uses

Edible or non-edible part

References

As spiny plant for Fencing and Hedge Fruit

Fruit is edible

[32] [12] [22] [30] [11] [33] [28] [23]

Fruit and root Root bark The plant is used as; fodder, wood for fuel, fruit yielding plant, used as timber, utilized for making furniture and agriculture appliances as well as plant for fence purposes. Leaves are browsed by goats. Spiny branches are used in fencing the fields. Wood, leaves, root, fruit. Wood used as fodder, fuel wood. Fruit, Root, Bark Roots

– Fruit is edible

Leaves



ethnomedicinal uses. The knowledge is folklore and transferred and preserved through literature review. Current review article supports this fact of folklore knowledge preservation. Though ZO has been part of various traditional treatment systems (Table 11), the ailment treated most widely with ZO are, its uses in hypertension, diabetes and jaundice. As mentioned in the table, the more widely used part of the plant for these purposes is root and root bark. Various parts of this plant are used traditionally as remedy for pain, diabetes, allergy, fever, rheumatism and pain [44] as well as in Ayurveda for treating fever, urinary troubles, skin infections and diabetes [45]. 3.11. Phytochemistry The phytochemical isolation from ZO yielded different compounds, most of which are reported for the first time and belongs to the class of phenolic, flavonoids and alkaloids. The dominant chemical class as observed is alkaloids particularly cyclopeptide alkaloids which consist of neutral, 13- and 14- membered rings as discussed in detail below. 3.11.1. Identification of phenolic compounds via HPLC-ESI–MS/MS analysis Qayum et al., studied the leaves, stem and fruit crude extract of ZO for chemical profiling. Although no actual isolation resulted in this study however different compounds from phenolic class were characterized and reported through HPLC-ESI–MS/MS techniques

[27] [14] [20] [34] [37] [36] [38]



[43]. The mass techniques consisted of ESI with [MH] mode. Hexosides of caffeic acid, ferulic acid, vanillic acid, sinapic acid as well as Kaempferol rutinoside were identified for different part of the plants. Fruit being the more enriched with phenolic compounds showed variety of phenolic compounds as shown in Table 12. The chromatograms for fruit (Fig. 3) and leaves (Fig. 4) crude extracts of ZO shows the presence of caffeic acid and sinapic acid with respective loss of hexose moieties i.e. (341 >179) and (385 > 223). 3.11.2. Cyclopeptide alkaloids Cyclopeptide alkaloids are basic, polyamidic compounds enormously distributed in various families i.e. Euphorbiaceae, Asteraceae, Sterculiaceae, Menispermaceae, Urticaceae, Rubiaceae, Celastraceae as well as Pandaceae however the most important and dominant class known for cyclopeptide alkaloids is the family Rhamnaceae and genus Ziziphus. On the basis of structure cyclopeptide alkaloids may be classified as 13-, 14- or 15membered as well as neutral cyclopeptide alkaloids. Apart from neutral cyclopeptide alkaloids, the aforementioned cyclopeptide classes contains 13-, 14- or 15- membered macrocycle in their structure formed due to presence of a styrylamine and a b-hydroxy-amino acid. These cyclopeptide alkaloids are further distinguished on the basis of a side chain with one (4 building blocks) or two (5 building blocks) additional amino acid attached on the ring, hence resulting in 4(13), 4(14), 5(13) and 5(14) Cyclopeptide subclasses. The neutral class of cyclopeptide alkaloids contains a cinnamoyl moiety instead of the basic amino

Table 11 Traditional/Ethnomedicinal uses of Ziziphus oxyphylla and its plant parts. Traditional uses

Part utilized for specific use References

Antidiabetic Curing jaundice Curing hypertension Curing jaundice Medicinal purposes Curing jaundice Used in gas trouble Hypertension, fever, aches, anti-dandruff, intestinal worms, pimples and bleeding gums Expulsion of intestinal worms Pimples and bleeding gums treatment Curing jaundice and hypertension Evil eye Digestive disorders, weakness, liver complaints, obesity, urinary troubles, diabetes, skin infections, fever, diarrhea and insomnia. Diabetes and Jaundice.

Fruit Roots Root bark Roots Whole plant Grinded roots Leaves Fruit, roots and barks Roots Roots Shrub, root Leaves Plant

[12] [11] [28] [33] [23] [27] [14] [20] [34] [36] [37] [38] [21]

Leaves and roots

[31]

978

R. Ahmad et al. / Biomedicine & Pharmacotherapy 91 (2017) 970–998

Table 12 Phenolic compounds profiling as present in Ziziphus oxyphylla fruit, leaves and stem. Extract

[M-H](m/ z) Z.O fruit Z.O leaves Z.O stem

Phenolic compounds Caffeic acid

Coumaric acidhexosides

Caffeic acidhexosides

Ferulic acidhexosides

Vanillic acidhexosides

Sinapic acidhexosides

Kaemferol-rutinoside

179

325

341

355

329

385

593

+  

+ + +

+  +

  

+ + +

 + +

+ + 

Fig. 3. MRM chromatograms of caffeic acid-hexosides. It is possible to note at least three isomers (at 15.51, 17.71 and 18.24 min), identified through the loss of hexose moiety (341 >179) and further fragmentation of caffeic acid (179 > 135).

Fig. 4. MRM chromatograms of sinapic acid-hexosides. It is possible to note at least two isomers (at 19.46 and 21.48 min), identified through the loss of hexose moiety (385 > 223) and further fragmentation of sinapic acid (223 > 149).

acid in the side chain. This class of cyclopeptide alkaloids is very rare and have been reported recently, for the first time in ZO [46]. 3.11.2.1. 13-Membered cyclopeptide alkaloids. A 13-membered cyclopeptide alkaloid i.e. Nummularine-R have been reported for the first time [47] from the stem methanol crude extract of ZO. Similarly another study [48] reported five (05) cyclopeptide alkaloids from root chloroform fraction of ZO methanolic extract with the addition of two (02) new 14-membered cyclopeptide alkaloids alongwith three already known 13-membered cyclopeptide alkaloids as; Oxyphylline-D (also known as hemsine), Nummularin-C, Nummularin-R. The more recent literature regarding isolation of cyclopeptide alkaloids reports nine (09) cyclopeptide alkaloids from root chloroform fraction of ZO methanol crude extract, among which

three (03) 13-membered cyclopeptide alkaloids; Nummularine-R and its two new derivatives O-desmethylnummularine-R and Odesmethylnummularine-R N-oxide and ramosine-A [46] as shown in Table 13. 3.11.2.2. 14-Membered cyclopeptide alkaloids. Alongwith 13membered cyclopeptide alkaloids, as mentioned earlier, 14membered cyclopeptide alkaloids too are reported from ZO. Inayat-Ur-Rahman et al. reported the isolation of a new 14membered cyclopeptide alkaloid i.e. Oxyphylline-A, for the first time in ZO stem methanol crude extract [47]. Two more, first time reported 14-membered cyclopeptide alkaloids; oxyphylline-B and oxyphylline-C were identified in root chloroform fraction of ZO methanol crude extract [48]. The more recent 14-membered cyclopeptide alkaloids isolated from root chloroform fraction of ZO methanol crude extract, are

R. Ahmad et al. / Biomedicine & Pharmacotherapy 91 (2017) 970–998

979

Table 13 Chemical group, plant part studied, and chemical constituents isolated from Ziziphus oxyphylla. Part used

Nature of extract used

Major chemical constituent

Representative class

References

Roots

Chloroform fraction of methanolic extract

Nummularine-R

13-membered cyclopeptide alkaloids

[46]

O-desmethylnummularine-R O-desmethylnummularine-R N-oxide Hemsine-A Ramosine-A Hemsine-A N-oxide Oxyphylline-C Oxyphylline-E Oxyphylline-F

14-membered cyclopeptide alkaloids

Neutral Alkaloids

Leaves

Ethyl acetate fraction of methanolic extract

Kaempferol-3-O-galactoside (Trifolin), Kaempferol-3-O-rhamnosyl (1–6)-(4”-trans-p- coumaroyl)galactoside, Quercetin-3-O–glucoside (Isoquercetrin) Kaempferol-3-O-glucosyl (1,2) rutinoside.

Flavonoids

[49]

Stem

Methanol

Nummularin-R

[47]

Chloroform fraction of methanolic extract

Oxyphylline-A Oxyphylline-B Oxyphylline-C Nummularin-R

13-membered cyclopeptide alkaloids 14-membered cyclopeptide alkaloids

Root

Aerial parts

[48]

13-membered cyclopeptide alkaloids

Oxyphylline-D Nummularin-C

Ethyl acetate fraction of methanolic extract

Kushecarpin-A

previously reported compounds i.e. hemsine-A and its derivative hemsine-A N-oxide and ramosine-A [46] as shown in Table 13. 3.11.2.3. Neutral cyclopeptide alkaloids. As aforementioned, this is a rare class of cyclopeptide alkaloids and no compound belonging to the class of neutral cyclopeptide alkaloids have been reported until now. Tuenter et al., reported for the first time three (03) new neutral cyclopeptide alkaloids compounds from root chloroform fraction of ZO methanol crude extract i.e. Oxyphylline-C, Oxyphylline-E and Oxyphylline-F as shown in Table 13 [46]. 3.11.3. Flavonoids Flavonoids, is a class of polyphenolic compounds with diverse chemical structure. The subclasses from flavonoids such as flavones, isoflavones, flavanones, flavonols, anthocyanins, catechins and chalcones are known for their potent antioxidant and radical scavenging activities. Although various phenolic compounds have been characterized and identified in ZO fruits, leaves and stem crude extracts [43] through HPLC-ESI–MS/MS however none of the studies have reported the isolation and structure elucidation of flavonoids from this plant. Ahmad et al., 2016a reported four (04) Kaempferol and Quercetin flavonoids from leaves ethyl acetate fraction of ZO methanol crude extract i.e. Kaempferol-3-O-galactoside (Trifolin), Kaempferol-3-O-rhamnosyl-(1–6)-(400 -trans-p-coumaroyl)-galactoside, Quercetin-3-O— glucoside (Isoquercetin) and Kaempferol-3-O-glucosyl (1,2) rutinoside. Although the flavonoids isolated hereby are known one however the isolation of such compounds from ZO is reported for the first time as shown in Table 13 [49]. Kushecarpin-A, primarily owe antibacterial and antiandrogen properties [50], was reported from the aerial part ethyl acetate fraction of the ZO methanol extract for the first time with potent anti-inflammatory activity [51].

Flavonoids

[51]

3.12. Structures of bioassay-guided isolated compounds from Ziziphus oxyphylla i) Alkaloids

Oxyphylline-A [47]

980

R. Ahmad et al. / Biomedicine & Pharmacotherapy 91 (2017) 970–998

Oxyphylline-D [47,48,52]

Oxyphylline-C [48,47]

Kuscecarpin-A [51]

Nummularin-C [47,48,52]

(1, 2, 3) Nummularin-R [47,48,52]

Oxyphylline-B [48]

(4, 5)

R. Ahmad et al. / Biomedicine & Pharmacotherapy 91 (2017) 970–998

(6)

981

(9) Nummularine-R (1), O-desmethylnummularine-R (2), O-desmethylnummularine-R N-oxide (3), Hemsine-A (4), Ramosine-A (6), Hemsine-A N-oxide (5), Oxyphylline-C (7), Oxyphylline-E (8) and oxyphylline-F (9) [46]. ii)Flavonoids

i) Kaempferol-3-O-galactoside (Trifolin) [49] (7)

ii) Kaempferol-3-O-rhamnosyl (1–6)-(4”-trans-p-coumaroyl)galactoside [49] (8)

982

R. Ahmad et al. / Biomedicine & Pharmacotherapy 91 (2017) 970–998

These studies supports the antioxidant role for this medicinal plant being used by local inhabitants for various ailments i.e. diabetes, jaundice, Alzheimer disease etc. [11,14,27,38,31].

iii) Quercetin-3-O–glucoside (Isoquercetrin) [49]

iv) Kaempferol-3-O-glucosyl (1,2) rutinoside [49] 3.13. Pharmacological activities

3.13.1.2. Total antioxidant (ABTS+) activity. Mazhar et al. measured the total antioxidant activity for the extract as well as fractions of ZO whole plant using phosphomolybednum method [41]. The results showed highest ABTS activity for chloroform fraction (1.723  0.34) whereas n-hexane (0.187  0.02) and aqueous fractions (0.138  0.13) exhibited less activity as compared to reference drug BHT (0.96  0.06). The ABTS+ radical activity, for ZO roots and leaves crude extracts, showed good activity for roots (IC50 = 0.73  0.04 mg/ml) as compared to leaves (1.39  0.07 mg/ml) crude extract [42]. In a follow up study [49], bioassay guided-isolated and tested four flavonoids for total antioxidant activity i.e. Kaempferol-3-Ogalactoside (Trifolin), Kaempferol-3-O-rhamnosyl-(1–6)-(4”transp-coumaroyl)-galactoside, Quercetin-3-O-glucoside (Isoquercetrin) and Kaempferol-3-O-glucosyl (1,2) rutinoside. The flavonoid with potent total antioxidant activity found, similar to the standard drug quercetin, was Quercetin-3-O-glucoside (180  5 mg/ml) (p = 0.05) as shown in Table 14. The total antioxidant activity is attributed due to the presence of phenols and flavonoids in the plant which support in part the antioxidant based folklore use of ZO in diabetes and jaundice [11,14,27,38,31]. 3.13.1.3. Superoxide (PMS-NADH) activity. Similarly free radical i.e. superoxide, scavenging activity was measured through PMS-NADH system. The roots and leaves methanol crude extract of ZO as tested [42] exhibited superoxide inhibitory activity for roots and leaves as 0.70  0.04 and 1.42  0.11 mg/ml as shown in Table 14. All these antioxidant, free radical as well as superoxide radical scavenging activities suggests the presence of phenols and flavonoids in ZO.

3.13.1. Antioxidant activity 3.13.1.1. DPPH. The methanol crude extract for ZO (whole plant) and its fractions in solvents of different polarities was studied [41]. The chloroform fraction revealed good antioxidant activity with DPPH% inhibition (95.01  0.37) and IC50 = 13.07  0.27 mg/ml when compared to the standard drug i.e. butylated hydroxytoluene (BHT) with IC50 = 12.10  0.29 mg/ml. Ahmad et al. studied the methanolic crude extract of ZO roots and leaves for% radical scavenging activity using different dilutions [40]. The potent free radical scavenging activity was observed for roots i.e. 97.8% and leaves i.e. 96.0% at dose concentration of 5  101 [40]. The result at mentioned dose concentration as compared to standard drug ascorbic acid revealed a significant free radical scavenging activity whereas increased dilution decreased this activity. In another study [53], the fractions from methanolic crude extract of ZO roots and leaves were studied for DPPH activity where a significant antioxidant (radical scavenging) activity was observed for the leaves ethyl acetate fractions (IC50 < 3 mg/ml) similar to quercetin (IC50 < 3.6 mg/ml) (P = 0.05). Ahmad et al., used semi preparative HPLC to further explore the leaves ethyl acetate fraction with isolation of four compounds (flavonoids) i.e. Kaempferol-3-O-galactoside (Trifolin), Kaempferol-3-O-rhamnosyl (1–6)-(4”-trans-p-coumaroyl)-galactoside, Quercetin-3-O-glucoside (Isoquercetrin) and Kaempferol-3-Oglucosyl (1,2) rutinoside [49]. Among isolated compounds, Quercetin-3-O-glucoside showed highest DPPH scavenging activity (IC50: 10.8  0.7 mg/ml). It was suggested that presence of more hydroxyl group imparts high antioxidant activity to quercetin glycosides as compared to Kaempferol glycosides as shown in Table 14.

3.13.2. Analgesic and antinociceptive activity Different pharmacological tests are available for evaluation of the analgesic and antinociceptive activity in animal models. ZO have been reported for analgesic and antinociceptive activity using the noxious chemicals induced procedures i.e. acetic acid induced writhing and formalin induced flinching behavior as discussed below; 3.13.2.1. Acetic acid-induced abdominal writhing. Nisar et al., reported first time the antinociceptive effects for methanolic extract of aerial parts from ZO [6]. The methanol crude extract for aerial parts of the plant at doses of 50, 100 and 200 mg/kg, in animal models induced with abdominal constriction using acetic acid, showed a dose dependent inhibition of the constrictions (p < 0.05). These effects were significant as compared to control drug i.e. Analgin [6]. Kaleem et al., isolated cyclopeptide alkaloids i.e. Oxyphylline-B, Oxyphylline-C, Oxyphylline-D, Nummularin-C, Nummularin-R from methanol crude extract of ZO with successive evaluation for acetic acid induced writhing inhibition [54]. At dose of 2.5 mg/ kg, ip., oxyphylline-B showed potent activity (57%) followed by nummularin-R (52.38%) whereas at dose of 5 mg/kg, both the compounds revealed a promising result in terms of amelioration of induced pains i.e. 80.98% and 77.87% respectively, showing a dose dependent activity for tested compounds. The study supports the antinociceptive activity as reported [6]. In another study [55], ZO plant crude extract was evaluated for acetic acid-induced writhing activity whereby a dose dependent analgesic effect was observed. The promising results were observed at higher doses i.e. 400 (83.79%) and 200 (72.76%) mg/ kg, comparative to standard drug aspirin i.e. 150 mg/kg (40.51%) p.

Table 14 Summary of the pharmacological activities for Ziziphus oxyphylla. Model used and study design Type of extract/isolated compound/s

Acetyl choline esterase inhibitory activity

Whole plant

Analgesic activity

Stem

Analgesic activity

Stem

In-vitro Acetylcholine esterase (AChE) inhibitory activity was measured by Ellman method. The reaction mixture (phosphate buffer, acetylcholine esterase, different concentrations of extract/fractions solution, DTNB) was incubated for 15 min and the hydrolysis of acetylthiocholine was monitored at 412 nm. Acetic Acid-Induced Abdominal Writhing test was performed on male mice (Swiss albino), weighting 19– 26 g. Five groups (six mice in group) were administered with aqueous solution of acetic acid (0.7%) and results were observed. Formalin-Induced analgesia was produced in Swiss albino mice grouped with six animals in each five groups. Injection of 0.05 ml of formalin in plantar surface of right hind paw was carried and the results were observed.

Antibacterial activity

Stem

Agar-well diffusion method using Muller-Hinton agar was applied for the study. The antibacterial activity was determined by measuring zones of inhibition of each sample wells. Both gram positive and gram negative bacteria such as Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, Bacillus subtilus and Shigella flexneri were used to evaluate the antibacterial activity of extract. Antibacterial activity was determined by the agar well diffusion technique using six pathogenic bacteria i.e. S. aureus, B. subtilis, P. mirabilis, S. typhi, E. coli and Citrobacter. The antibacterial activity was

3 mg/ml Methanolic extract and its fractions i.e. n-Hexane, chloroform, ethyl acetate, nButanol and aqueous fraction

Leaves

Stem and leaves

Root and leaves

Tested dose/s

Positive control

Possible mechanism of action Observations

30–150 mg/ml Methanolic extract and its fractions i.e. n-Hexane, chloroform, ethyl acetate, nButanol and aqueous fraction

Galanthamine

The AChE activity is due to the presence of high amount of phenolic, flavonoids, alkaloids and terpenoids content.

The n- butanol fraction showed lowest IC50 value (9.58  0.08 mg/ml), indicating that it contained the best inhibition of the enzyme relative to galanthamine (13.26  0.73 mg/ml)

[41]

Methanolic extract

100, 200 and 400 mg/kg

Aspirin

The analgesic activity of extract is due to antiinflammatory effects as in visceral pain model arachidonic acid releases cyclooxygenase responsible for nociceptive mechanism.

Crude Extract of ZO showed analgesic effects in a dose dependent manner against writhing induced by acetic acid. The more potent affect observed was at dose of 400 and 200 mg/kg comparative to Aspirin’s effect.

[55]

Methanolic extract

50, 100 and 200 mg/kg

Aspirin

The analgesic activity was observed due to inhibitory effect on direct activation of sensory neurons and central sensitization of sensory neurons in the second phase by decreasing the licking time and frequency.

ZO crude extract with doses ranging from 50 mg/kg up to 200 mg/kg, ip. significantly inhibited both early and late phase of pain stimulus. The analgesic effect was significant (P < 0.05) in dose of 200 mg/kg.

[55]

Imipenem

Specific compounds present in plant extracts may contribute to treatment of infection disorder like fungal and bacterial diseases. No clear mechanism for these activities was mentioned.

Neither crude extract nor any [58] of subsequent fractions showed any antibacterial activity. Ethyl acetate fraction showed [44] good activity (16 mm zone of inhibition) against B. subtilus and (18 mm zone of inhibition) S. aureusus whereas remaining fraction along with crude extract did not show any inhibition. [56] The result revealed that oil sub-fractions did not show any inhibition.

Neomycin and doxycycline The higher antibacterial activities for roots may owe to the higher amounts of total phenols as previous studies have reported that the inhibition of growth of microorganisms could be

Dose dependent activity was [42] observed. Roots crude extract exhibited significantly higher zone of inhibition of 22.29  0.04 mm against S. aureus whereas the leaves crude extract showed

Oil sub-fractions WO-1 to WO-4 (stem oil) and WO-5 (oil from leaves) Methanolic extract

10–1500 mg/ ml

References

983

Part/s Used

R. Ahmad et al. / Biomedicine & Pharmacotherapy 91 (2017) 970–998

Pharmacological Activity

984

Table 14 (Continued) Pharmacological Activity

Part/s Used

Model used and study design Type of extract/isolated compound/s

Tested dose/s

Positive control

Antifungal activity

Leaves and roots

Stem

considerable activity at doses higher than 300 mg/ml. The highest inhibitory activity for leaves was against S. typhi (22.15  0.01 mm). The antibacterial activity of Z. All the fractions from leaves [53] oxyphylla may be due to the and root crude extract were inactive against E. coli presence of cyclopeptide whereas the only fractions alkaloids as this class of active against S. aureus were compounds have well chloroform as well as ethyl established antibacterial acetate fraction with an IC50 activity reported. value; 15.5 and 22.4 ug/ml respectively.

The antibacterial activity was carried out for E. coli and S. aureus using Mueller–Hinton broth. The inoculum consisted of 105 CFU/mL for bacteria. Results were interpreted spectrophotometrically by plate reader after addition of MTT and resazurin as redox indicator The isolated compounds were screened for antibacterial activity against E. coli, B. subtilis, S. flexeneri, S. aureus, P. aeruginosa and S. typhi using disc diffusion technique. A known volume (10 ml) of the solution was applied to sterilized filter paper discs and dried at room temperature. The plates were incubated at 37  C for 12–15 h and results recorded as zone of inhibition (mm).

Fractions from methanolic extract i.e. n-Hexane, chloroform, ethyl acetate, nButanol and aqueous fraction

Dilutions i.e. 64, 16, 4, 1, 0.25 mg/ml from stock solutions (20 mg/ml).

Chloramphenicol (E. coli) and Erythromycin (S. aureus)

Cyclopeptide alkaloids; Oxyphylline-B (4) Oxyphylline-D 1 Nummularin-C (2) Nummularin-R (3) Oxyphylline-C (5)

10 mg/ml

Imipenem

The investigation of the bacterial molecular target can facilitate the knowledge of its molecular mechanism of action.

Oxyphylline-B showed [48] comparatively better antibacterial activities against E. coli (MIC, 5 mg/ml) as well as weak antimicrobial activities against S. aureus (MIC, 25 mg/ml) P. aeruginosa (MIC, 50 mg/ml) and S. typhi (MIC, 50 mg/ml). OxyphyllineD, nummularin-C, nummularin-R and oxyphylline-C exhibited low antibacterial activities.

The antifungal activity was carried out for T. rubrum, A. fumigatus and C. albicans using Sabouraud broth. The inoculum consisted of 104 CFU/mL for fungi. Results were interpreted by plate reader after addition of MTT and resazurin as redox indicator. The antifungal activity was determined by Agar tube dilution method. Six fungi Trichophyton longifusis, Candida albicans, Candida glabarata, Fusarium solani, Microsporum canis and Aspergillus flavus were used to see the antifungal activity. The samples were incubated for 7 days at 29C and growth inhibition was observed. The

Fractions from methanolic extract i.e. n-Hexane, chloroform, ethyl acetate, nButanol and aqueous fraction

Dilutions i.e. 64, 16, 4, 1, 0.25 mg/ml from stock solutions (20 mg/ml).

Miconazole



[53] None of the fraction were active against any of the fungal strain tested. The plant shows no antifungal activity.

Miconazole and amphotericin-B

Specific compounds present in plant extracts may contribute to treatment of infection disorder like fungal and bacterial diseases. No clear mechanism for these activities was mentioned.

Maximum antifungal activity [58] (35%) was shown against M. canis by n-hexane fraction followed by crude extract and ethyl acetate fraction with antifungal activity of 30%. The n-butanol fraction exhibited 20% antifungal activity whereas crude extract and aqueous fraction showed 10% inhibition against A. flavus and F. solani respectively. No activity was showed against T.

400 mg/ml Methanolic extract and its fractions i.e. n-Hexane, chloroform, ethyl acetate, nButanol and aqueous fraction

R. Ahmad et al. / Biomedicine & Pharmacotherapy 91 (2017) 970–998

Stem

References

attributed to phenolic compounds present in the extracts.

determined by measuring zones of inhibition of each sample wells.

Leaves and roots

Possible mechanism of action Observations

percentage growth inhibition was calculated. Leaves

Stem and leaves

Anti-glycation activity

Chloroform fraction of stem and root methanolic extract Root and leaves

Leaves

Leaves

Anti-inflammatory activity

Stem

Oil sub-fractions WO-1 to WO-4 (stem oil) and WO-5 (oil from leaves)

In-vitro BSA-MGO assay was performed and each sample was examined for the development of specific fluorescence (excitation, 330 nm; emission, 440 nm), against blank.

Cyclopeptide alkaloids, i) nummularine-R ii) nummularin-C iii) hemsine-A



Methanolic extract

Aminoguanidine 200 ml test compounds of different concentrations

Rutin

Compounds 1 and 3 showed antiglycation potential with IC50; 720.2 and 277.7 mM, respectively.

The high phenolic and flavonoid content may be the probable reasons for antiglycation activity.

Leaves methanolic extract [42] showed considerably high IC50 value (1.32  0.04) compared to roots crude extract (1.41  0.04 mg/ml). The result showed a [49] significant activity with IC50 values of 0.60 mg/ml for ethyl acetate fraction.

Hyperglycation is considered to increase oxidative stress. Glycation and oxidation appear to be linked hence flavonoid glycosides with a role as AGEs inhibitors have been proved here.

Fractions from methanolic extract i.e. n-Hexane, chloroform, ethyl acetate, nButanol and aqueous fraction Flavonoids; 1. Kaempferol-3-Ogalactoside (Trifolin) 2. Kaempferol-3-Orhamnosyl (1–6)-(4”-transp- coumaroyl)-galactoside 3. Quercetin-3-O–glucoside (Isoquercetrin) 4. Kaempferol-3-O-glucosyl (1,2) rutinoside.

50, 200 and 400 mg/kg

Indomethacin

Inflammatory mediators i.e. prostaglandins, cyclooxygenase and kinins were reduced.

[45]

All the compounds except compound 2 (IC50; 530 mg/ ml) revealed comparatively same antiglycation activity.

[49]

The overall activity of drug was really stronger as compare to saline and standard drug Indomethacin throughout and was found influential in a dose dependent manner. The curative time for first two hour at 400 mg/kg drug concentration was found

[55]

985

Methanolic extract Male mice (Swiss albino), weighting 19–26 g were used in the study and carrageenaninduced edema model was used where acute inflammation was produced by sub planter injection of 0.1 ml of 1% suspension of carrageenan in normal saline, in the hind paw of the mice. 1 h after the oral



R. Ahmad et al. / Biomedicine & Pharmacotherapy 91 (2017) 970–998

castaneum, S. oryzea and T. granarium. Maximum antifungal activity [44] (35%) was shown against M. canis by crude, n-hexane and aqueous fraction whereas chloroform and butanol fraction showed 30% and 20% activity, respectively. Ethyl acetate and n-hexane fraction showed low activity against A. flavus (10% to 20%). Maximum antifungal activity [56] (35%) was shown against M. canis by WO-1 and WO-2 whereas no inhibitory effect of oil sub-fractions was seen against remaining fungal strains.

986

Table 14 (Continued) Pharmacological Activity

Part/s Used

Model used and study design Type of extract/isolated compound/s

Tested dose/s

Positive control

Possible mechanism of action Observations significant in treating inflammatory disorders.

administration of the test sample as well as the positive and negative controls the paw volume was measured plethysmometrically Leaves

References

Kushecarpin-A

5, 10 and 20 mg/kg



[58] Carrageenan-induced paw edema was significantly reduced in a dose-dependent fashion by the treatment of Kushecarpin-A.

Leaves and roots

L. infantum inoculum was prepared, by harvesting the infected donor hamster’s spleen amastigote and used to infect murine peritoneal macrophages. 104 cells/ well of the peritoneal cells were added to microtiter plate, incubated, prediluted extracts added and after 5 days the parasitic burden was noted through microscope using Giemsa stain. Results were expressed as percentages.

Fractions from methanolic extract i.e. n-Hexane, chloroform, ethyl acetate, nButanol and aqueous fraction

Dilutions i.e. 64, 16, 4, 1, 0.25 mg/ml from stock solutions (20 mg/ml).

Miltefosine

The antileishmanial activity of Z. oxyphylla may be sue to the presence of cyclopeptide alkaloids as this class of compounds have well established activity reported.

The only fraction active [53] against L. infantum inhibition was root chloroform fraction with IC50 value, 32.4 ug/ml. all other fractions were inactive.

Antinociceptive activity

Dichloromethane fraction of methanolic root extract

Formalin induced flinching behavior test was performed by injecting 0.05 ml of formalin (2.5%) into the plantar surface of the right hind paw, 30 min after treating the animals with the compounds. Nociceptive behavior was quantified as the animal walking or can stand on the injected paw. For analgesic activity, Acetic acid induced writhing test was performed using BALB/c mice (18–22 g) of either sex. After 30 min of the treatment with normal saline and diclofenac, the animals were injected with acetic acid (1% ip.). The abdominal constriction (writhing) was counted for 10 min after 5 min of acetic acid injection

Cyclopeptide alkaloids; i. Oxyphylline-B ii. Oxyphylline-C iii. Oxyphylline-D iv. Nummularin-C v. Nummularin-R

2.5 and 5 mg/ kg

Tramadol

The strong antinociceptive effect in the post-formalin induced flinching behavior test may be attributed to different mechanisms i.e. peripheral as well as central effects of the compounds.

[54] Compound 1 provoked 45.32% and 75.32% while compound 5, 36.77% and 71.10% protection in the 1st and 2nd phases of formalin induced flinching behavior test respectively

2.5 and 5 mg/ kg

Diclofenac

The possible mechanism of the antinociceptive activity of these compounds could be due to the blockade of the effect or the release of endogenous substances (arachidonic acid metabolites) that excite pain nerve endings.

The compounds antagonized abdominal constrictions in a dose dependent manner at 2.5 and 5 mg/kg ip. At the dose of 2.5 mg/kg ip., maximum pain alleviation (57%) was observed for compound 1 followed by compound 5 (52.38%). At 5 mg/kg ip., both the compounds demonstrated a promising boost in pain amelioration.

50, 100 and 200 mg/kg

Analgin

This pain mechanism is believed to involve, in part,

Aerial parts of the For antinociceptive activity plant acetic acid-induced writhing

Methanolic extract

[54]

[6]

R. Ahmad et al. / Biomedicine & Pharmacotherapy 91 (2017) 970–998

Antileishmanial activity

was performed over Swiss albino mice (20–28 g) of both sexes. Briefly, 10 ml/kg of 0.7% aqueous solution of acetic acid were injected into mice (ip.) 30 min after treatment with saline, extract or Analgin. Abdominal constriction i.e. contortions of the abdominal muscle (stretching of hind limps) that occurred between 5 and 15 min after acetic acid injection were counted and expressed as percent inhibition of nociception

Leaves and roots

Roots

Antipyretic activity Stem

Fractions from methanolic extract i.e. n-Hexane, chloroform, ethyl acetate, nButanol and aqueous fraction

Dilutions i.e. 64, 16, 4, 1, 0.25 mg/ml from stock solutions (20 mg/ml).

Chloroquine

Nine (09) isolated Cyclopeptide alkaloids; Nummularine-R (1), Odesmethylnummularine-R (2), Odesmethylnummularine-R N-oxide (3), Hemsine-A (4), Ramosine-A (6), Hemsine-A N-oxide (5), Oxyphylline-C (7), Oxyphylline-E (8) and oxyphylline-F (9)

Yeast-induced pyrexia model Methanolic extract was used. Pyrexia was induced in Male rats, weighting 170–220 g, by a sub-cutaneous injection of 10 ml/kg of 15% water solution of yeast. Adult Wistar rats (180–260 g) were injected 10 ml/kg s.c. of 15% aqueous solution of yeast. The rectal temperature of each animal was again recorded at 0.5, 1, 1.5 and 2 h after treatment. Antipyretic effect was rated as the ability of test articles to reverse the induced pyrexia.

50, 200 and 400 mg/kg

For T. brucei trypomastigote were grown in HMI-9

Dilutions i.e. 64, 16, 4, 1,

Fractions from methanolic extract i.e. n-Hexane,

Aspirin

Paracetamol

Suramine

The antiplasmodial activity of Z. oxyphylla may be sue to the presence of cyclopeptide alkaloids as this class of compounds have well established antiplasmodial activity reported. It was concluded that cyclopeptide alkaloids with a 2-hydroxystyrylamine moiety, like compound 2 and terminal cinnamoyl moiety and tryptophane unit present in compound oxyphylline-F (9) are responsible for antiplasmodial activity.

The highest inhibitory activity [53] was observed for chloroform fraction of leaves and roots i.e. 8.8 and 13.2 mg/ml.

Nummularine-R (1), Odesmethylnummularine-R (2) and oxyphylline-F (9) showed the highest antiplasmodial activity (IC50 values of 3.2, 7.1 and 7.4 mM respectively).

[46]

Doses of 200 and 400 mg/kg [55] showed maximum deduction in elevated rectal temperature of rats as compare to 50 mg/kg dose indicates the potential effect of extract of Z. oxyphylla. [6] The result showed that 100 The extract caused a and 200 mg/kg of the extract significant hypothermal activity against yeast-induced significantly (p < 0.05) pyrexia via encounter of the reversed hyperpyrexia in rats. synthesis of prostaglandin. The potential effect was observed due to decrease in prostaglandins.

The antitrypanosomal The fractions exhibiting activity of Z. oxyphylla may be considerable inhibition

[53]

987

Leaves and roots

Resistant strain of P. falciparum (K1 strain), cultured in RPMI-1640 medium supplemented with 4% human erythrocytes and 10% human serum, was selected and maintained in microaerophilic culture as determined in procedure. Assay was performed in 96 well microtiter plate using phenazine ethosulfate (2 mg/ ml) and nitro blue tetrazolium (0.1 mg/ml) and the change in color was noted at 655 nm spectrophotometrically.

The abdominal constrictions induced by acetic acid were inhibited dose-dependently.

R. Ahmad et al. / Biomedicine & Pharmacotherapy 91 (2017) 970–998

Antiplasmodial activity

local peritoneal receptors caused by peritoneal fluid concentrations of PGE2 and PGF2a as relieved by plant extract.

Pharmacological Activity

Part/s Used

Antitrypanosomal activity (T. brucei)

Leaves and roots

Brine shrimp lethality assay

Roots and leaves

Stem

Leaves

Tested dose/s

medium, incubated at standard conditions followed by addition of 104 parasites in each microtiter well. After 4 days incubation period, resazurin was added for assessment of growth of the parasite by fluorimetric method. Tulahuen strain of T. cruzi grown on MRC-5 cells, on minimal essential medium was dispensed in 96 well microtiter plate. Betagalactosidase substrate (chlorophenol red-b-Dgalactopyranoside) was added to well and color was examined at 540 nm for results.

chloroform, ethyl acetate, n- 0.25 mg/ml from stock Butanol and aqueous solutions fraction (20 mg/ml).

Eggs of brine shrimp Artemia salina were hatched in Sea salt (38 g/L in water and maintained the pH at 7.4). incubation at 37  C lead to hatching in 2 days and 10 larvae/vial were taken and added up with samples. The samples were illuminated under light for 24 h and the results taken as the number of larvae survived processed through Finney computer programme to find the LD50 value. Artificial “sea water” was prepared and filtered, placed in a small tank; added brineshrimp eggs (1 mg) (Artemia salina) and was darkened by covering with aluminum foil. Then 10 shrimps were added per vial, allowed to stand for 24 h, shrimps were counted and recorded the number of surviving shrimps. Artificial “sea water” was prepared and filtered, placed in a small tank; added brineshrimp eggs (1 mg) (Artemia salina) and was darkened by covering with aluminum foil. Then 10 shrimps were added per vial, allowed to stand for 24 h, shrimps were counted and recorded the number of surviving shrimps.

Methanolic extract

Positive control

Possible mechanism of action Observations sue to the presence of cyclopeptide alkaloids.

Benznidazole

Fractions from methanolic extract i.e. n-Hexane, chloroform, ethyl acetate, nButanol and aqueous fraction

100, 10 and 5ug/ml

20 mg/ Methanolic extract and its 2 ml fractions i.e. n-Hexane, chloroform, ethyl acetate, nButanol and aqueous fraction

References

against T. brucei were chloroform fraction of both, root and leaves crude extract with IC50 value of 21.1 and 23.5 ug/ml, respectively.

For T. cruzi the more potent [53] inhibition was shown by chloroform fraction of the root crude extract (IC50; 1.84 ug/ml) followed by the IC50 value; 19.7 ug/ml (leaves chloroform fraction). However the n-Hexane fraction of the leaves also revealed considerable results with IC50 value; 22.9 ug/ml.

Cyclophosphamide

The presence of potent chemical classes of different compounds is the reason behind its activity.

[40] The root methanolic extract showed high cytotoxicity even at low doses i.e. 5 ug/ml with LD50 value of 45.74 ug/ ml.

Etoposide



[58] Results also showed that neither crude extract nor any of the fractions showed any cytotoxic activity.



[44]

R. Ahmad et al. / Biomedicine & Pharmacotherapy 91 (2017) 970–998

Antitrypanosomal activity (T. cruzi)

Model used and study design Type of extract/isolated compound/s

988

Table 14 (Continued)

Artificial “sea water” was prepared and filtered, placed in a small tank; added brineshrimp eggs (1 mg) (Artemia salina) and was darkened by covering with aluminum foil. Then 10 shrimps were added per vial, allowed to stand for 24 h, shrimps were counted and recorded the number of surviving shrimps.

Oil sub-fractions WO-1 to WO-4 (stem oil) and WO-5 (oil from leaves)



No cytotoxic effects were observed.

[56]

Cytotoxicity assay

Chloroform fraction of stem and root methanolic extract Leaves and roots

In-vitro cytotoxicity of the compounds was assayed by MTT assay using PC-3 cell line. The absorbance reading was taken at 570 nm. MRC-5 cells were cultivated in minimum essential medium provided followed by suspension of 104 MRC-5 cells in each well of micro plate containing pre-diluted plant extract. Plate was incubated at specified conditions and followed by addition of resazurin. Fluorescence was measured as excitation 550 nm, emission 590 nm, and the results were interpreted as% reduction in cell viability of extract treated well compared to control

Cyclopeptide alkaloids, i) nummularine-R ii) nummularin-C iii) hemsine-A



Doxorubicin



All of these compounds failed [45] to show growth inhibition of 20% at 50 mM.

Fractions from methanolic extract i.e. n-Hexane, chloroform, ethyl acetate, nButanol and aqueous fraction

Dilutions i.e. 64, 16, 4, 1, 0.25 mg/ml from stock solutions (20 mg/ml).

Tamoxifen

The chloroform and ethyl [53] acetate fraction of root as well as chloroform fraction of leaves revealed cytotoxicity with IC50 values as; 5.1, 19.99, and 18.22 ug/ml, respectively.

Nine (09) isolated Cyclopeptide alkaloids; Nummularine-R (1), Odesmethylnummularine-R (2), Odesmethylnummularine-R N-oxide (3), Hemsine-A (4), Ramosine-A (6), Hemsine-A N-oxide (5), Oxyphylline-C (7), Oxyphylline-E (8) and oxyphylline-F (9)

Tamoxifen

The cytotoxicity is well known property of the presence of alkaloids as present in chloroform fractions and hence the only active fraction was chloroform. –

In-vitro DPPH radical scavenging activity of crude extract and fractions were examined by addition of different concentration test samples with 3 ml of methanolic DPPH solution (0.1 mM). The absorbance (517 nm) was measured and

15  1000 mg/ Methanolic extract and its fractions i.e. n-Hexane, ml chloroform, ethyl acetate, nButanol and aqueous fraction

BHT The DPPH radical (butylatedhydroxytoluene) scavenging is thought to be due to the hydrogen donating ability and presence of high amount of phenolic, flavonoids, alkaloids and terpenoids content.

Roots

DPPH (free radical scavenging activity)

Whole plant

IC50 value higher than 64.0 mM was found for compound 2 i.e. Odesmethylnummularine-R during cytotoxicity studies.

[46]

R. Ahmad et al. / Biomedicine & Pharmacotherapy 91 (2017) 970–998

Stem and leaves

Chloroform fraction showed [41] the highest percent inhibition of DPPH radical (95.01  0.37) with IC50 value 13.07  0.27 mg/ml followed by n-Butanol fraction 29.79  1.30 mg/ml.

989

990

Table 14 (Continued) Pharmacological Activity

Part/s Used

Roots and leaves

Leaves

result was interpreted as IC50 value. The DPPH (80 mg/ml) solution was prepared by dissolving 4 mg in 50 ml of ethanol. Different dilutions of the crude extract i.e. 5  102, 5  103, 5  104, 5  105, 5  106, 5  107, 5  108, 5  109, 5  1010 mg/ml were prepared and 1 ml of the dilution was mixed with 1 ml DPPH. The absorbance at 517 nm was measured and result as% inhibition was calculated. In-vitro DPPH scavenging activity of the isolated compounds was determined via addition of test compound of different concentrations in ethanol to 1.0 ml of the ethanolic DPPH. The percent inhibition was calculated at 517 nm.

Leaves

FRAP (Ferric reducing antioxidant power) Assay

Whole plant

Tested dose/s

Positive control

Possible mechanism of action Observations

Methanolic extract

10 mg/ml

Ascorbic acid

The presence of potent chemical classes of different compounds is the reason behind its activity.

[40] Both the methanolic extract i.e. root and leaves, showed a very good and potent antioxidant activity however roots were very potent amongst all the dilutions tested for these plants. The highest dilution and lowest dilution resulted a percent inhibition of 61.87 and 97.86% respectively.

Fractions from methanolic extract i.e. n-Hexane, chloroform, ethyl acetate, nButanol and aqueous fraction

0.5 ml of different concentration samples

Quercetin

The isolated compounds exhibited different antioxidant activities due to the number of hydroxyl groups present in the aromatic ring. The isolated compounds exhibited different antioxidant activities due to the number of hydroxyl groups present in the aromatic ring. Quercetin glycosides have better antioxidative properties than Kaempferol glycosides, which may be due to the presence of a higher number of hydroxyl groups in the aromatic ring.

The ethyl acetate fractions [53] showed high DPPH scavenging activity and accordingly low IC50 values of 3.0 mg/ml as compared to other fractions. E3 and E4 sub-fractions [49] exhibited comparatively high radical scavenging activity i.e. IC50 (0.08 mg/ml).

High FRAP values obtained from polar fractions showed the presence of flavonoid and phenolic contents. Phenolic and flavonoid contents show significant antioxidant action on human health and fitness. These compounds/ antioxidants act through scavenging or chelating process. The high potential of phenolic to scavenge free radicals may possibly be due to various phenolic hydroxyl groups they possess.

Chloroform fraction showed [41] highest FRAP value (339.5  0.57 TE mM/mL). Ethyl acetate fraction also showed good FRAP value i.e. 296.01  0.85 TEmM/mL, while n-hexane and remaining aqueous fractions showed very less FRAP values i.e. 21.50  0.19 TEmM/mL and 107.3  0.64 TEmM/mL, respectively.

Four sub-fractions (E1, E2, E3, E4) of ethyl acetate fraction from methanolic extract

Flavonoids; 1. Kaempferol-3-Ogalactoside (Trifolin) 2. Kaempferol-3-Orhamnosyl (1–6)-(4”-transp- coumaroyl)-galactoside 3. Quercetin-3-O–glucoside (Isoquercetrin) 4. Kaempferol-3-O-glucosyl (1,2) rutinoside.

FRAP solution was prepared by mixing 25 ml acetate buffer (pH 3.6), 2.5 ml TPTZ solution, 2.5 ml ferric chloride hexahydrate solution. The solutions of plant samples and that of trolox were prepared in methanol (500 mM/ml). 50 ml of each fraction was taken in separate test tubes and 150 ml of FRAP solution was added in each. The absorbance of the colored product was checked at 593 nm.

50 ml of each Methanolic extract and its fractions i.e. n-Hexane, fraction chloroform, ethyl acetate, nButanol and aqueous fraction

Trolox

Compound 3 exhibited the highest DPPH scavenging activity (IC50: 10.8 mg/ml).

References

[49]

R. Ahmad et al. / Biomedicine & Pharmacotherapy 91 (2017) 970–998

Leaves

Model used and study design Type of extract/isolated compound/s

Insecticidal activity

Stem

Crude extract and all fractions 200 mg/3 ml were evaluated against different insect’s viz., Tribolium castaneum, Callosbruchus analis, and Rhyzopertha dominica. The results were analyzed as percentage mortality, calculated with reference to the positive and negative controls.

Permethrin

[58] Crude along with other fraction were inactive against T. castaneum, S. oryzea and T. granarium.



Stem and leaves

Methanolic extract

The method consisted of insects Tribolium castaneum and Rhyzopertha Dominica loaded on a petri dish with paper containing the dissolved samples (samples in volatile oil kept for 24 h to dry). Ten (10) insects were placed in the petri dish and the results were observed after 24 h for survival as% inhibition of mortality.

Leaves

Spectroscopic method using Kushecarpin-A lipoxygenase (EC 0.13.11.12) type I-B (Soybean) and linoleic acid was used to determine lipoxygenase inhibitory activity. The reaction was initiated by the addition of 10 ml linoleic acid substrate solution and the absorption change with the formation of (9Z,11E)-13S)13-hydroperoxyoctadeca-

10 ml of sample solution

Baicalein



Kushecarpin-A exhibited significant inhibition of soybean lipoxygenase having the IC50 value 7.2  0.02 mM.

[51]

991

Roots and leaves

R. Ahmad et al. / Biomedicine & Pharmacotherapy 91 (2017) 970–998

The result obtained indicated [44] that n-hexane and ethyl acetate fraction showed moderate insecticidal activity (40%) against C. analis whereas other fractions accept crude showed nonsignificant (20%) activity. Crude extract showed nonsignificant activity against R. dominica. Crude along with other fraction were inactive against T. castaneum, S. oryzea and T. granarium. WO-2 showed nonsignificant [56] Oil sub-fractions WO-1 to – activity while WO-1 and WOWO-4 (stem oil) and WO-5 5 revealed moderate and good (oil from leaves) insecticidal activity against R. dominica and T. castaneum respectively. Oil sub-fractions remained inactive against C. analis. [40] No insecticidal activity was – The presence of potent chemical classes of different reported for any of the root or leave methanolic extract. compounds is the reason behind its activity.

Leaves

Lipoxygenase inhibition



992

Table 14 (Continued) Pharmacological Activity

Part/s Used

Model used and study design Type of extract/isolated compound/s

Tested dose/s

Positive control

Possible mechanism of action Observations

Paraquat

Phytotoxic activity is dose dependent i.e. high phytotoxcicity at high concentrations and viceversa.

Quercetin

The results indicate that the antioxidant activity of the extracts is related with the total phenol content.

References

9,11-dienoate was followed for 10 min.

Phytotoxic activity

Stem

Phytotoxic activity was determined using Lemna minor. The number of fronds per flask were counted and recorded on day seven and their growth regulation in percentage was calculated.

20 mg/ml Methanolic extract and its fractions i.e. n-Hexane, chloroform, ethyl acetate, nButanol and aqueous fraction

SuperoxideRadical scavenging activity

Stem and leaves

Oil sub-fractions WO-1 to WO-4 (stem oil) and WO-5 (oil from leaves)

Root and leaves

Methanolic extract PMS-NADH System was applied for superoxide radical scavenging ability of extracts. The absorbance was measured after 5 min in 560 nm. The superoxide radical scavenging activity was calculated as% radical Flavonoids; scavenging. 1. Kaempferol-3-Ogalactoside (Trifolin) 2. Kaempferol-3-Orhamnosyl (1–6)-(4”-transp- coumaroyl)-galactoside

Leaves

0.1 ml different concentration samples

The PMS superoxide radical [42] inhibition, exhibited significant scavenging activity with IC50 of 0.70  0.04 and 1.42  0.11 mg/ml for root and leaves extract respectively. The compound 3 showed Quercetin glycosides have [49] significant superoxide better antioxidative scavenging activity with IC50 properties than Kaempferol glycosides, which may be due value of 400 mg/ml. to the presence of a higher number of

R. Ahmad et al. / Biomedicine & Pharmacotherapy 91 (2017) 970–998

Leaves

Crude as well as other fraction [58] showed significant activity at highest concentration (1000 mg/ml), maximum activity was shown by crude extract at this concentration that is 90% growth regulation while 60% was the lowest activity as shown by aqueous fraction. Crude as well as other fraction [44] showed significant activity at highest concentration (1000 mg/ml), maximum activity was shown by crude extract at this concentration that is 90% growth regulation while 60% was the lowest activity as shown by aqueous fraction. [56] WO-2 and WO-5 fraction showed significant activity at highest concentration (1000 mg/ml), maximum activity was shown by WO-2 and WO-5 at this concentration that is 80% and 70.59% growth regulation while 15% and 23.13% was the lowest activity by same fractions. Similarly WO-1 at this concentration showed 45% growth regulation while 25% was the lowest activity by same fraction.

Total Antioxidant Activity (ABTS. + assay)

Root and leaves

Leaves

a-Chymotrypsin inhibitory activity

a-Glucosidase inhibitory activity

Chloroform fraction of stem and root methanolic extract

Flavonoids; 1. Kaempferol-3-Ogalactoside (Trifolin) 2. Kaempferol-3-Orhamnosyl (1–6)-(4”-transp- coumaroyl)-galactoside 3. Quercetin-3-O–glucoside (Isoquercetrin) 4. Kaempferol-3-O-glucosyl (1,2) rutinoside. 500 mg of each BHT Methanolic extract and its Phosphomolybdenum fractions i.e. n-Hexane, fraction (butylatedhydroxytoluene) complex formation method was used. Brief; 500 mg/ml of chloroform, ethyl acetate, neach fraction was mixed with Butanol and aqueous fraction 4 ml of reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate), incubated at 95oC for 90 min successively followed by absorbance measurement at 695 nm.

In-vitro inhibitory activity of chymotrypsin was performed in 50 mM Tris–HCl buffer with 10 mM CaCl2, pH 7.6. Change in absorbance was continuously monitored at 410 nm. a-Glucosidase inhibitory activity was assayed by using 0.1 M phosphate buffer (pH 6.8) at 37 8C and change in absorbance at 400 nm was monitored up to 30 min.

Cyclopeptide alkaloids; i) nummularine-R ii) nummularin-C iii)hemsine-A

The results indicate that the antioxidant activity of the extracts is related with the total phenol content.

The ABTS+ radical scavenging activity showed IC50 values for roots and leaves methanolic extract as; 0.72  0.04 and 1.39  0.07 mg/ml.

[42]

Quercetin glycosides have better antioxidative properties than Kaempferol glycosides, which may be due to the presence of a higher number of hydroxyl groups in the aromatic ring.

The total antioxidant activity measured as ABTS radical scavenging ability showed that compounds 3 had (IC50; 170 mg/ml) similar results to quercetin (180 mg/ml).

[49]

The total antioxidant activity may be attributed due to presence of large amount of phenolic, flavonoids, alkaloids and terpenoids content.

[41] The chloroform fraction showed highest total antioxidant activity (1.723  0.34) while the nhexane and remaining aqueous fractions exhibited very less activity (0.187  0.02 and 0.138  0.13 respectively)

No compound exhibited any [45] inhibitory effect against a-chymotrypsin enzyme hence these compounds are unlikely to interfere with the digestion of proteins in the food. All cyclopeptide alkaloids 1– [45] 3, were found to inhibit a-glucosidase enzyme. Compound 1 showed IC50 = 212.0 mM, compound 2 possess IC50 = 251.0 mM, while compound 3 showed IC50 = 394.0 mM.



Chymostatin





1-Deoxynojirimycin



R. Ahmad et al. / Biomedicine & Pharmacotherapy 91 (2017) 970–998

Whole plant

The total antioxidant activity of extracts was measured by the ABTS.+ method, where ABTS.+ radical cation was generated. The percentage decrease of the absorbance at 734 nm was calculated. The total antioxidant activity of extracts was measured by the ABTS+ method, where ABTS+ radical cation was generated. The percentage decrease of the absorbance at 734 nm was calculated.

hydroxyl groups in the aromatic ring.

3. Quercetin-3-O–glucoside (Isoquercetrin) 4. Kaempferol-3-O-glucosyl (1,2) rutinoside. Methanolic extract

993

994

R. Ahmad et al. / Biomedicine & Pharmacotherapy 91 (2017) 970–998

o. The significant results (P < 0.05) as observed for ZO crude extract supports the previous reports as shown in Table 14. 3.13.2.2. Formalin induced flinching behavior. Five cyclopeptide alkaloids i.e. Oxyphylline-B, Oxyphylline-C, Oxyphylline-D, Nummularin-C, Nummularin-R have been isolated and tested for inhibitory effects in post formalin induced flinched animal models [54]. All the compounds revealed a dose dependent reduction in formalin induced flinching behavior however oxyphylline-B (45.32% and 55.10%) and nummularin-R (37.77% and 50.10%) were more effective in attenuation of noxious stimuli at dose of 2.5 mg/kg, ip., in both phases. At dose of 5 mg/kg, same compounds were dominant again with activity in 1st (neurogenic) and 2nd (tonic) phase respectively as 45.32% and 75.32% for oxyphylline-B and 36.77% and 71.10% for nummularin-R. Similarly, Ali et al., studied ZO crude extract in dose range of 50– 200 mg/kg, ip., whereby a significant (P < 0.05) analgesic effect was observed at dose concentration of 200 mg/kg [55]. The study is in concordance with the previous report [54], however the standard drug Aspirin exhibited strong activity at the tested dose as shown in Table 14. These studies supports the fact; ZO is used traditionally to treat fever, inflammation and pain [21,56]. 3.13.3. Antipyretic activity The methanol extract of aerial part i.e. leaves from ZO was studied for antipyretic potential in in-vivo animal models at doses of 100 and 200 mg/kg. The hyperpyrexia was significantly reduced by the extract (p < 0.05) however less effective as compared to standard drug Paracetamol [6]. A similar study was reported [55], where the rats (induced with pyrexia using yeast) treated with ZO crude extract at doses of 50, 100, 200 and 400 mg/kg showed a significant dose dependent decrease in rectal hyperpyrexia as shown in Table 14. The doses i.e. 200 and 400 mg/kg in particular revealed a maximum activity as compared to other doses. Aspirin was used as standard drug. These studies are in agreement for the antipyretic and thus folkloric uses of ZO in traditional system of medicines. These studies supports the fact; ZO is used traditionally to treat fever, inflammation and pain [21,56]. The genus species is famous for its analgesic, anti-inflammatory and pain reducing potential as reported [38,7,57]. 3.13.4. Anti-inflammatory activity The crude extract as well as isolated compound from ZO plant have been tested for anti-inflammatory activity in in vivo rats model as reported [58,55]. Both studies used Carrageenan-induced paw edema model. A new compound from ZO i.e. Kushecarpin-A was isolated and evaluated for anti-inflammatory activity at doses of 5, 10 and 20 mg/kg. All the tested doses exhibited significant (p < 0.05) as well as high significant (p < 0.01) results, showing a potent activity [51]. Whereas the study [55], also revealed a worth mentioning activity in the aforementioned animal model at doses of 50, 100 and 200 mg/kg. The most influential dose observed in reducing the hind paw inflammation was 400 followed by 200 mg/kg as shown in Table 14. The activity was dose dependent and comparatively stronger than standard drug indomethacin. These studies supports the fact; ZO is used traditionally to treat fever, inflammation and pain [21,56]. The genus Ziziphus is famous for its analgesic, anti-inflammatory and pain reducing potential as reported [38,7,57]. 3.13.5. Antiglycation activity Advanced glycation end products, a complex group of compounds have been implicated in the pathogenesis of diabetes,

neurodegenerative diseases, atherosclerosis and Alzheimer’s disease. Since hyperglycation is considered to increase oxidative stress, glycation and oxidation appear to be inextricably linked [59]. AGEs such as glyoxal, methyl glyoxal, fructosamine etc. are formed due to combination of proteins and sugars in the body which leads to further diabetic complications. The AGEs prevention by ZO extracts have been studied and reported as below; Choudhary et al., isolated cyclopeptide alkaloids i.e. Nummularin-R and C and Hemsine-A and studied its antiglycation potential using bovine serum albumin model [45]. NummularinR and hemisne-C were found active against protein glycation with IC50 values as 720.2  10.9 mM and 277.7  7.6 mM respectively. This report is in concordance with previous study [60], where inhibition of protein glycation was shown due to presence of high phenolic contents in plants. Ahmad et al., in another study tested the leaves and roots crude extract of ZO for protein glycation end products inhibition [42]. The results demonstrated a highest activity for leaves crude extract (1.41  0.04 mg/ml) followed by root crude extract (1.32  0.04 mg/ ml) which is again in line with previous reports [60,45]. Furthermore, based on high antioxidant and protein glycation inhibition properties, leaves ethyl acetate fraction (due to high phenolic and flavonoid contents) was further subjected to active isolation [49]. Four flavonoids glycosides i.e. Kaempferol-3-Ogalactoside (Trifolin), Kaempferol-3-O-rhamnosyl(1–6)-(4”-transp-coumaroyl)-galactoside, Quercetin-3-O—glucoside (Isoquercetin) and Kaempferol-3-O-glucosyl (1,2) rutinoside were isolated and the ability to inhibit protein glycation was evaluated using BSA-glucose assay. All the isolated compounds showed low IC50 values for protein glycation inhibition ranging from 530 to 818 mg/ ml, comparable to Aminoguanidine (510 mg/ml) used as a positive control. Isoquercetin, among the isolated flavonoid glycosides, showed a highest inhibitory activity i.e. 530  19 mg/ml as shown in Table 14. This study too, is consistent with previous reports [60,45]. The potent inhibitory activity as AGEs inhibitor supports the folklore use of this plant in diabetes and diabetes related complications as reported [12,61,21,31]. 3.13.6. Antidiabetic property a -Glucosidase, is an enzyme which is secreted from intestinal epithelium. The main role of this enzyme is to catalyze the conversion of polysaccharides into monosaccharide hence providing source for glucose. However in diabetic patients the mentioned conversion as well as enzymatic activity needs to be inhibited in order to decrease the amount of free glucose in blood which may lead to diabetic complications. Thus inhibition of a -glucosidase have an indirect role in decreasing the risks of hyperglycemia in diabetic patients. Choudhary et al., in a study on ZO plant isolated Cyclopeptide alkaloids i.e. Nummularin-R and C and Hemsine-A and further studied its a -glucosidase enzyme inhibitory activity [45]. All of the compounds were found inhibitory for this enzyme with IC50 values as 212.0  1.6 mM, 251.0  1.2 mM and 394.0  2.4 mM respectively. The results observed established a good role for these compounds as antidiabetic agents via inhibition of the a -glucosidase enzyme activity as shown in Table 14. The a -glucosidase enzyme inhibitory activity supports the folklore uses of the plant in diabetes and its related complications as reported [12,61,21,31]. 3.13.7. Antibacterial activity Various plant parts as well as extract in different polarity solvents for ZO have been reported for antibacterial activity against Gram (+)ve as well as Gram ()ve bacterial strains. The first antibacterial study for ZO leaves methanolic crude extract and its

R. Ahmad et al. / Biomedicine & Pharmacotherapy 91 (2017) 970–998

fractions was carried out [44], using agar well diffusion method for both G (+)ve and G ()ve species i.e. Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, Bacillus subtilus and Shigella flexneri. None of the fractions except ethyl acetate showed any antibacterial activity. Ethyl acetate fraction was the more potent, only against B. subtilis and S. aureus with antibacterial activity measured as zone of inhibition i.e. 16 and 18 mm respectively. In a similar study [58], ZO stem methanolic extract and its fractions were studied for antibacterial activity against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, Bacillus subtilus and Shigella flexneri using agar well diffusion method. The crude extract as well as fractions showed no activity against any of the tested pathogens. The oil extracted from leaves and stem methanolic extract of ZO was tested for antibacterial activity against G (+)ve and G ()ve strains i.e. Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, Bacillus subtilus and Shigella flexneri using agar well diffusion method. No antibacterial activity was observed for oil sub-fraction against any of the tested bacterial strains [56]. The results from this study are in concordance with previous investigation [62]. Kaleem et al., isolated 13- and 14-membered cyclopeptide alkaloids from chloroform fraction of ZO methanolic extract and evaluated further for antibacterial activity using disc diffusion method for bacterial strains including Escherichia coli, Bacillus subtilis, Shigella flexeneri, Staphylococcus aureus, Pseudomonas aeruginosa and Salmonella typhi [48]. All the isolated cyclopeptide alkaloids exhibited a weak antibacterial activity against tested pathogens however Oxyphylline-B revealed a comparatively better activity against E. coli and S. aureus with minimum inhibitory concentration of 5 and 50 mg/ml, respectively. Other compounds i.e. Oxyphylline-D, Nummularin-C as well as Nummularin-R showed negligible activity against tested pathogens. Furthermore, Ahmad et al., 2013, tested the antibacterial potency of the roots and leaves crude methanol extracts of ZO against various species of bacteria including the Gram-positive bacteria S. aureus, B. subtilis and the Gram-negative bacteria P. mirabilis, S. typhi, E. coli and Citrobacter using agar well diffusion method [42]. Root crude extract showed significant results at small dose concentration of 10–150 mg/ml. The most interesting results were against S. aureus (22.29  0.04 mm) at dose concentration of 150 mg/ml whereas, moderate results were observed for all other bacteria tested i.e. S. typhi (19.86  0.06 mm), Citrobacter (18.29  0.05 mm), E. coli (17.62  0.03), P .mirabilis (17.21  0.05 mm) and B. subtilis (17.00  0.05 mm). The extract also showed activity at small dose concentration of 10 mg/ml, except against S. aureus, P. mirabilis and S .typhi where the extract was ineffective inhibiting these bacteria at dose of 10 mg/ml. Leaves crude extract showed no activity at doses up to 150 mg/ml whereby an increase in dose above 300 mg/ ml i.e. up to 1500 mg/ml, revealed considerable activity against S. typhi (22.15  0.01 mm), E. coli (20.42  0.02 mm) and Citrobacter (19.13  0.05 mm) as shown in Table 14. Standard drug used was doxycycline. Although the result for current study was according to the findings of [56,44] however a difference in result was observed compared to report by [58]. In another study Ahmad et al., reported the antibacterial activity for different polarity fraction (Chloroform, ethyl acetate, butanol, residual methanol) of ZO roots and leaves crude extract however the method i.e. serial dilution method using microtiter plate was different [53]. The only significant activity observed was against S. aureus whereas none of the leaves or roots fraction was effective against E. coli at doses up to 64 mg/ml. The fraction effective against S. aureus was chloroform and ethyl acetate fraction from roots with IC50 of 15.5 and 22.4 mg/ml, respectively and chloroform fraction from leaves with IC50 54.8 mg/ml. These results are in accordance to previous antibacterial studies [44,58].

995

3.13.8. Anti-fungal activities Nisar et al., reported the first time any antifungal activity for ZO leaves crude methanolic extract and its fractions i.e. n-hexane, chloroform, ethyl acetate and butanol against fungi i.e. Candida albicans, Microsporum canis, Trichophyton longifusis, Aspergillus flavus, Fusarium solani and Candida glabarata [44]. The results observed showed; maximum activity i.e. 35% for n-hexane and aqueous fraction against M. canis, moderate activity i.e. 30% and 20% for chloroform and butanol fraction respectively against M. canis, whereas a low activity i.e. 10% and 20% was shown against A. flavus from ethyl acetate and n-hexane fractions. Similarly another study [58], reported antifungal activity for ZO stem crude extract and its fractions i.e. n-hexane, chloroform, ethyl acetate and butanol against Fusarium solani, Microsporum canis, Candida albicans, Aspergillus flavus and Candida glabarata. Like previous study [44], maximum activity i.e. 35% was observed for n-hexane against M. canis. Crude extract as well as ethyl acetate fraction exhibited maximum activity i.e. 30% followed by n-butanol fraction with an activity of 20% against M. canis. The inhibition against F. solani and A. flavus observed was very less i.e. 10% from crude extract and aqueous fraction. On contrary to the previous studies, Kaleem et al., reported no antifungal activity for the sub-fractions of ZO oil fraction i.e. WO-1 and WO-2 against tested pathogens i.e. Candida albicans, Fusarium solani, Trichophyton longifusis, Microsporum canis, Aspergillus flavus and Candida glabarata. However the fractions itself i.e. WO-1 and WO-2 revealed a maximum activity of 35% against M. canis [56]. Ahmad et al., tested nine different pathogenic fungi, Y. aldovae, A. parasiticus, C. albicans, A. niger, S. cerevisiae, F. solani, T. rubrum, A. effuses and M. phaseolina for antifungal potency of the leaves and root crude extract of Z. oxyphylla [42]. Agar well diffusion method was used. No activity was observed for any of the crude extract at any of the tested dose of 10–150 mg/ml as shown in Table 14. These results were in compliance with the already reported antifungal activity on Z. oxyphylla plant as [48] [56,44]. Ahmad et al., in another antifungal study, using different methodology i.e. serial dilution method in microtiter plate against A. fumigatus, T. rubrum and C. albicans for ZO leaves and roots crude extract and its fractions reported no antifungal activity for any of the fractions [53]. This result is similar to previous studies [44,58]. 3.13.9. Toxicity studies Toxicity study is very important in order to know the pharmacological activities of a plant crude extract as well as its therapeutic index. Different in-vitro studies have been reported as follows; 3.13.9.1. Brine shrimp activity. Nisar et al., Kaleem et al. and Ahmad et al., evaluated ZO stems, leaves and roots crude extract and its oil sub-fractions on brine shrimps i.e. Artemisia salina at various dose concentrations (5, 10 and 100 mg/ml). Results in term of LD50 were calculated. All these studies reported lack of cytotoxicity for any of the tested crude extract or oil sub-fractions of ZO except the presence of cytotoxicity in roots crude extract [40], as shown in Table 14. This cytotoxicity is suggested to be due to presence of alkaloids in the root crude extract. 3.13.9.2. Insecticidal activity. Nisar et al. [44,58], studied the insecticidal activity of leaves and stem crude extract and its fractions against Tribolium castaneum, Sitophilus oryzea and Trogoderma granarium. None of the fraction as well as crude extract from ZO leaves showed any insecticidal activity against the tested insects whereas in case of stem crude extracts and fractions, ethyl acetate and n-hexane fraction showed moderate activity (40%) against C. analis. The remaining fractions were inactive against any of the tested insects.

996

R. Ahmad et al. / Biomedicine & Pharmacotherapy 91 (2017) 970–998

Another study [56], also reported lack of significant insecticidal activity for oil sub-fractions isolated from ZO plant against the tested insects i.e. Rhyzopertha dominica and Tribolium castaneum. Ahmad et al., 2014, reported no insecticidal activity for ZO leaves and bark methanolic crude extract against Tribolium castaneum and Rhyzopertha dominica as shown in Table 14 [40]. This study is in line with the previous reports [44,51,56]. 3.13.9.3. Cytotoxicity studies on MRC-5 cell lines. Ahmad et al., 2016b, reported MRC-5 cell line toxicity for ZO leaves and root crude extract and its fractions using microtiter plate serial dilution method [53]. The chloroform fraction of root and leaves crude extract were the more cytotoxic against these cell line as shown in Table 14. This study is in concordance with previous report [40], where brine shrimp cytotoxicity is reported for root crude extract. 3.13.10. Phytotoxicity studies Besides aforementioned toxicity studies, phytotoxicity evaluation was also performed for ZO crude extract and its fractions. Nisar et al., studied phytotoxicity for ZO leaves crude extract and its fractions against Lamina minor [58]. All the tested samples showed phytotoxicity at higher doses only i.e. 1000 mg/ml where the highest (90%) and lowest (60%) activity of growth regulation was exhibited by crude extract and aqueous fraction, respectively. In another similar study for ZO stem crude extract and its fractions [51], it was reported that phytotoxicity for this ZO plant crude extract and its fraction is dose dependent i.e. at higher doses (1000 mg/ml) a maximum activity was observed as shown in Table 14. The study supports the previous reports [44]. Kaleem et al., reported the phytotoxicity study for oil and its sub-fractions (WO1-WO5) obtained from ZO plant [56]. Here again a dose dependent activity i.e. maximum activity at higher doses i.e. 1000 mg/ml was observed for all the tested samples. Sub-fractions WO-2 and WO-5 showed a growth regulation of 80% and 70.59%, respectively at higher doses whereas at lower doses the growth regulation observed was 15% and 23.13%, respectively. 3.13.11. Antileishmanial activity Ahmad et al., studied the anti-infective activity of ZO leaves and roots methanolic crude extract and its fractions i.e. n-hexane, ethyl acetate, chloroform and n-butanol against Leishmania infantum in serial dilution method using microtiter plate [53]. The results revealed ZO roots chloroform fraction with highest inhibitory activity i.e. IC50: 32.4 mg/ml as compared to standard drug miltefosine (IC50: 10.41 mM) as shown in Table 14. This is first time report for any anti-infective study on this plant. These results supports the anti-infective traditional uses of plant as reported [34]. 3.13.12. Antiplasmodial activity The crude extract and its fractions, as mentioned in antileishmanial study, for ZO leaves and roots, were also evaluated for its anti-plasmodia activity against the resistant K-1 strain of Plasmodium falciparum using serial dilution method [53]. The chloroform fraction of ZO roots and leaves showed effective inhibition against malarial parasite with IC50 value of 5.25 and 8.8 mg/ml, respectively. Ethyl acetate (IC50: 13.2 mg/ml) and nhexane (IC50: 24.1 mg/ml) also revealed anti-plasmodia activity however it was lower as compared to standard drug chloroquine (IC50: 0.35 mM). No previous anti-plasmodia activity has been reported for this plant as shown in Table 14. These results supports the anti-infective traditional uses of plant as reported [34]. 3.13.13. Antitrypanosomal activity The crude methanol extract and its fractions i.e. n-hexane, ethyl acetate, chloroform and n-butanol were also studied against two

trypanosome strains i.e. T. cruzi and T. brcei. Almost all of the fractions were active against T. cruzi in the IC50 range of 6.06–22.9 mg/ ml, however root chloroform fraction (1.84 mg/ml) was more active comparative to standard drug benzidazole (IC50: 2.09 mM) Furthermore, in the case of T. brucei again all the fractions were active from both leaves and root crude extract of ZO however root chloroform fraction (21.1 mg/ml) was more active followed by leaves chloroform and n-Hexane fraction with IC50 value of 23.5 and 29.7 mg/ml, respectively, as compared to standard drug suramine (IC50: 0.02 mM) as shown in Table 14 [53]. These results supports the anti-infective traditional uses of plant as reported [34]. 3.13.14. Acetyl choline esterase inhibitory activity Acetylthiocholine esterase (AChE) inhibition decreases the rate of acetyl choline (ACh) degradation and increases its concentration in the brain. Mazhar et al., 2015, studied the AChE inhibitory activity for ZO methanol crude extract and its fractions i.e. nhexane, ethyl acetate, chloroform, n-butanol etc. via spectrophotometric method [41]. n-butanol fraction was the more effective enzyme inhibitor at low IC50 value of 9.58  0.08 mg/ml as compared to standard drug galanthamine (13.26  0.73 mg/ml) as shown in Table 14. 3.13.15. Lipoxygenase (LOX) inhibitory activity Nisar et al., isolated and studied the LOX inhibitory activity of a flavonoid i.e. Kushecarpin-A. Significant results i.e. IC50 value of 7.2  0.02 mM were observed as compared to standard drug Baicalein (IC50: 22.0  0.05 mM) as shown in Table 14 [51]. LOX are responsible for fatty acids metabolism whereby inflammatory mediator are released hence resulting inflammation. LOX inhibitor are suggested as best agent to encounter these inflammatory mediators. The results for this study suggests a detail mechanism of compound isolated from Zo crude extract hence supporting the traditional uses of this plant as an anti-inflammatory and pain reducing as reported [38,7,57]. 4. Conclusion The research studies have shown that antioxidants protect against glycation-derived free radicals and may have therapeutic potential [63]. Furthermore, it is reported that compounds with combined antioxidant and antiglycation properties are more effective in treating diabetes mellitus [64]. The antioxidant activity as observed in the literature confirms the role and uses of ZO in traditional or folkloric disease of liver and jaundice [38] along with diabetes [12]. The isolated flavonoid glycosides with established antioxidant properties indicates possible applications as well as preventive measure for ZO against glycation-associated complications in diabetes. Cyclopeptide alkaloids as reported for the first time from ZO further confirms the anti-infective, hepatoprotective as well as antioxidant activity of the plant thus applicable in folkloric system of treatment. The outcomes of the reported studies regarding ZO may be useful in developing protocols for the effective use of the pharmaceutical and other biological properties of this plant in conventional system of medicine. This plant observed a great piece of work in terms of research during the last few years with recent literature reports. The crude extracts, fractions, sub-fractions as well as isolated compounds from Z. oxyphylla alongwith phytochemical, biological and toxicological studies confirms the role of these plants in its folkloric uses. Conflict of interest No conflict of interest exist among authors.

R. Ahmad et al. / Biomedicine & Pharmacotherapy 91 (2017) 970–998

Ethics approval and consent to participate Not applicable. Consent for publication No individual person’s data in any form is included in this review manuscript. Authors contribution RA, created the idea and proposed the research topic. NA and AAN performed the necessary literature survey and compiled the relevant data. RA, wrote the article whereas NA and AAN under the guidance of RA structured the tables and images. RA and NA arranged the references as per journal requirement. RA and AAN, reviewed the article for any linguistic or typographical mistakes. Funding source Not applicable. Acknowledgment Not applicable. References [1] D. Katiyar, V. Singh, M. Ali, Phytochemical and pharmacological profile of Pterocarpus marsupium: a review, Pharma Innov. J. 5 (4) (2016) 31–39. [2] A. Gosh, Herbal folk remedies of Bantura & Medinipur districts, West Bengal (India). Indian J Tradit Knowl 2, 393–396, Pak. J. Bot. (2003) 1445–1452. [3] A.O. Al-Zaher, S.Y. Salim, M.H. Assaf, R.H. Abdel-Hady, Antidiabetic activity and toxicity of Zizyphus spina-christi leaves, J. Ethnopharmacol. 101 (2005) 129– 138. [4] B.H. Han, M.H. Park, Folk Medicines: The Art and Science, The American Chemical Society, Washington, DC, 1986, pp. 205. [5] K.R. Kirtikar, B.D. Basu, Indian Medicinal Plants, Springer, Berlin, 1984, pp. 593. [6] M. Nisar, B. Adzu, K. Inamullah, A. Bashir, A. Ihsan, A.H. Gilani, Antinociceptive and antipyretic activities of the Ziziphus oxyphylla edgew leaves, Phytother. Res. 21 (2007) 693–695. [7] B. Adzu, S. Amos, C. Wambebe, K. Gamaniel, Antinociceptive activity of Zizyphus spina-christi root bark extract, Fitoterapia 72 (2001) 344–350. [8] S.M. Al-Reza, V.K. Bajpai, S.C. Kang, Antioxidant and antilisterial effect of seed essential oil and organic extracts from Zizyphus jujube, Food Chem. Toxicol. 47 (2009) 2374–2380. [9] M.C. De Omena, D.M.A.F. Navarro, J.E. De Paula, J.S. Luna, Larvicidal activities against Aedes aegypti of some Brazilian medicinal plants, Bioresour. Technol. 98 (2007) 2549–2556. [10] A. Perveen, M. Qaiser, Pollen flora of pakistan–xliv. Rhamnaceae, Pak. J. Bot. 37 (2) (2005) 195–202. [11] G. Jan, M.A. Khan, F. Gul, Ethnomedicinal plants used against Jaundice in Dir Kohistan valleys (NWFP), Pakistan, Ethnobot. Leafl. 13 (2009) 1029–1041. [12] H. Sher, Ethnoecological evaluation of some medicinal and aromatic plants of Kot Malakand Agency, Pakistan, Sci. Res. Essays 6 (2011) 2164–2173. [13] M.A. Khan, S.A. Khan, M.A. Qureshi, G. Ahmed, M.A. Khan, M. Hussain, G. Mujtaba, Ethnobotany of some useful plants of Poonch Valley Azad Kashmir, J. Med. Plants Res. 5 (2011) 6140–6151. [14] Z. Sher, Z.D. Khan, F. Hussain, Ethnobotanical studies of some plants of Chagharzai valley, district Buner, Pakistan, Pak. J. Bot. 43 (2011) 1445–1452. [15] A.M. Abbasi, M.A. Khan, M. Ahmad, M. Zafar, Medicinal Plants Biodiversity of Lesser Himalayas-Pakistan, vol. XIII(2012) 220 p. 116 illus. in. colour, Hardcover. (ISBN 978-1-4614-1575-6). [16] http://www.efloras.org/florataxon.aspx?flora_id=5&taxon_id=10763 & http:// www.tropicos.org/Image/100164455. [17] Edgew, Rhamnaceae., Ziziphus oxyphylla Edgew, Trans. Linn. Soc. Lond. 201 (1846) 43 [1851 publ. 29 Aug 1846]. [18] Bhandari, Bhansali, Rhamnaceae, Ziziphus oxyphylla Var. Pedicellaris, vol. 20, Fasc. Fl. India, 1990, pp. 108. [19] http://efloras.org/florataxon.aspx?flora_id=5&taxon_id=250075389. [20] M.P.Z. Khan, M. Ahmad, M. Zafar, S. Sultana, M.I. Ali, H. Sun, Ethnomedicinal uses of edible wild fruits (EWFs) in swat valley, northern Pakistan, J. Ethnopharmacol. 173 (2015) 191–203. [21] W.A. Kaleem, N. Muhammad, H. Khan, A. Rauf, Pharmacological and phytochemical studies of genus Zizyphus, Middle-East J. Sci. Res. 21 (8) (2014) 1243–1263.

997

[22] A. Jabeen, M.A. Khan, M. Ahmad, M. Zafar, F. Ahmad, Indigenous uses of economically important flora of Margallah Hills National Park, Islamabad, Pakistan, Afr. J. Biotechnol. 8 (5) (2009) 763–784. [23] U. Samreen, Ibrar, M. Badshah, L. Naveed, S. Imran, I. Khatak, Ethnobotanical study of subtropical hills of Darazinda, Takht-e-Suleman range F.R D.I. Khan, Pakistan, Pure Appl. Biol. 51 (2016) 149–164. [24] Z. Saqib, A. Sultan, Ethnobotany of Palas valley, Pakistan, Ethnobot. Leafl. 5 (2005) 1350–1357. [25] S. Sultana, H.M. Asif, N. Akhtar, K. Ahmad, Medicinal plants with potential antipyretic activity: a review, Asian Pac. J. Trop. Dis. 5 (Suppl. 1) (2015) S202– S208. [26] M. Ajaib, Z. Khan, N. Khan, M. Wahab, Ethnobotanical studies on useful shrubs of district Kotli, Azad Jammu and Kashmir, Pakistan, Pak. J. Bot. 42 (3) (2010) 1407–1415. [27] M.S. Amjad, Ethnobotanical proiling and loristic diversity of Bana Valley, Kotli (Azad Jammu and Kashmir), Pakistan, Asia. Pac. J. Trop. Biomed. 5 (4) (2015) 292–299. [28] M.A. Khan, M.A. Khan, M. Hussain, Medicinal plants used in folk recipes by the inhabitants of himalayan region Poonch valley Azad Kashmir (Pakistan), J. Basic Appl. Sci. 8 (2012) 35–45. [29] A. Shah, A. Karim, J. Ahmad, M.P. Sharma, Medicinal shrubs used by GujjarBakerwal tribes against various non-communicable diseases in Rajouri district, (J & K), India, Indian J. Tradit. Knowl. 14 (3) (2015) 466–473. [30] S. Kumar, I.A. Hamal, Ethnobot. Leafl. 13 (2009) 195–202. [31] M. Ajaib, M. Anjum, N.Z. Malik, M.F. Sidiqui, Ethnobotanical study of some plants of Darguti, tehsil Khuiratta, Azad Jammu and Kashmir, Int. J. Biol. Res. 3 (2) (2015) 101–107. [32] F.U. Haq, H. Ahmad, M. Alam, I. Ahmad, Rahatullah, Species diversity of vascular plants of Nandiar Valley Western Himalaya, Pakistan, Pak. J. Bot. 42 (2010) 213–229 (Special Issue (S.I. Ali Festschrift)). [33] R.A. Rafiq, The flora of Palas valley and plant conservation priorities. Report on the botanical studies in Palas valley (1992–1995), Report Prepared for Himalayan Jungle Project, Palas Valley, Kohistan, (1995) . [34] A.M. Abbasi, M.A. Khan, N. Khan, M.H. Shah, Ethnobotanical survey of medicinally important wild edible fruits species used by tribal communities of Lesser Himalayas-Pakistan, J. Ethnopharmacol. 148 (2013) 528–536. [35] M. Khan, F. Hussain, S. Musharaf, Ethnobotanical profile of Tehsil Takht-eNasratti, District Karak, Pakistan, J. Med. Plants Res. 7 (22) (2013) 1636–1651. [36] A.M. Abbasi, M.A. Khan, M. Ahmad, M. Zafar, S. Jahan, S. Sultana, Ethnopharmacological application of medicinal plants to cure skin diseases and in folk cosmetics among the tribal communities of North-West Frontier Province, Pakistan, J. Ethnopharmacol. 128 (2010) 322–335 s.n.. [37] S.A. Shah, N.A. Shah, S. Ullah, M.M. Alam, H. Badshah, S. Ullah, A.S. Mumtaz, Documenting the indigenous knowledge on medicinal flora from communities residing near Swat River (Suvastu) and in high mountainous areas in SwatPakistan, J. Ethnopharmacol. 182 (2016) 67–79 s.n.. [38] A.H. Shah, A.M. Ai-Bekairi, S. Qureshi, A.M. Agee, Zizyphus sativa fruits: evaluation of some biological activities and toxicity, Phytother. Res. 3 (2006) 232–236. [39] R. Niamat, M.A. Khan, K.Y. Khan, M. Ahmad, B. Ali, P. Mazari, M. Mustafa, H. Ahmad, Element content of some ethnomedicinal Ziziphus Linn. species using atomic absorption spectroscopy technique, J. Appl. Pharm. Sci. 2 (03) (2012). [40] R. Ahmad, M. Ahmad, J.N. Mehjabeen, Phytochemical screening and Antioxidant activity of the two plants Ziziphus oxyphylla Edgew and Cedrela serrata Royle, Pak J. Pharm. Sci. 27 (5) (2014) 1477–1482. [41] F. Mazhar, R. Khanum, M. Ajaib, M. Jahangir, Potent AChE enzyme inhibition activity of Zizyphus oxyphylla: a new source of antioxidant compounds, Pak J. Pharm. Sci. 28 (6) (2015) 2053–2059. [42] R. Ahmad, A. Upadhyay, M. Ahmad, L. Pieters, Antioxidant, antliglycation and antimicrobial activities of Ziziphus oxyphylla and Cedrela serrata extracts, Eur. J. Med. Plants 3 (4) (2013) 520–529. [43] M. Qayum, M. Zia-Ul-Haq, W.A. Kaleem, S. Ahmad, L. CALANI, T. Mazzeo, N. Pellegrini, Antioxidant potential of impatiens bicolor royle and Zizyphus oxyphylla edgew, Pak. J. Bot. 46 (5) (2014) 1725–1729. [44] M. Nisar, W.A. Kaleem, M. Qayum, A. Hussain, M. Zia-ul-Haq, I. Ali, M.I. Choudhary, Biological screening of Zizyphus oxyphylla Edgew leaves, Pak. J. Bot. 42 (2010) 4063–4069. [45] M.I. Choudhary, A. Adhikari, S. Rasheed, B.P. Marasini, N. Hussain, W.A. Kaleem, Atta-ur-Rahman, Cyclopeptide alkaloids of Ziziphus oxyphylla Edgew as novel inhibitors of a-glucosidase enzyme and protein glycation, Phytochem. Lett. 4 (4) (2011) 404–406 s.n.. [46] E. Tuenter, R. Ahmad, K. Foubert, A. Amin, M. Orfanoudaki, P. Cos, L. Maes, S. Apers, L. Pieters, V. Exarchou, Isolation and structure elucidation by LC-DADMS and LC-DAD-SPE-NMR of cyclopeptide alkaloids from roots of Ziziphus oxyphylla. and evaluation of antiplasmodial activity, J. Nat. Prod. 79 (2016) 2865–2872 s.n.. [47] M.A. Inayat-Ur-Rahman Khan, M. Arfan, G. Akhtar, L. Khan, V.U. Ahmad, A new 14-membered cyclopeptide alkaloid from Ziziphus oxyphylla, Nat. Prod. Res. 21 (2007) 243–253. [48] W.A. Kaleem, M. Nisar, M. Qayum, M. Zia-Ul-Haq, A. Adhikari, V.D. Feo, New 14-membered cyclopeptide alkaloids from Ziziphus oxyphylla edgew, Int. J. Mol. Sci. 13 (2012) 11520–11529. [49] R. Ahmad, N. Ahmad, A.A. Naqvi, V. Exarchou, A. Upadhyay, E. Tuenter, K. Foubert, S. Apers, N. Hermans, L. Pieters, Antioxidant and antiglycating constituents from leaves of Ziziphus oxyphylla and Cedrela serrata, Antioxidants 51 (2016) E9.

998

R. Ahmad et al. / Biomedicine & Pharmacotherapy 91 (2017) 970–998

[50] M. Kuroyanagi, T. Arakawa, Y. Hirayama, T. Hayashi, Antibacterial and antiandrogen flavonoids from sophora flavescens, J. Nat. Prod. 62 (12) (1999) 1595–1599. [51] M. Nisar, W.A. Kaleem, I. Khan, A. Adhikari, N. Khan, M.R. Shah, I.A. Khan, M. Qayum, M. Samiullah Ismail, A. Aman, Molecular simulations probing Kushecarpin A as a new lipoxygenase inhibitor, Fitoterapia 82 (2011) 1008– 1011. [52] M. Nisar, W.A. Kaleem, A. Adhikari, Z. Ali, N. Hussain, I. Khan, M. Qayum, M.I. Choudhary, Stereochemistry and NMR data assignment of cyclopeptide alkaloids from Ziziphus oxyphylla, Nat. Prod. Commun. 5 (2010) 1205–1208. [53] R. Ahmad, N. Ahmad, A.A. Naqvi, P. Cos, L. Maes, S. Apers, N. Hermans, L. Pieters, Anti-infective, cytotoxic and antioxidant activity of Ziziphus oxyphylla and Cedrela serrata, Asia. Pac. J. Trop. Biomed. 6 (8) (2016) 671–676. [54] W.A. Kaleem, N. Muhammad, M. Qayum, H. Khan, A. Khan, L. Aliberti, V. De Feo, Antinociceptive activity of cyclopeptide alkaloids isolated from Ziziphus oxyphylla Edgew (Rhamnaceae), Fitoterapia 91 (2013) 154–158. [55] R. Ali, H.U. Shah, I. Ullah, J. Anwar, M. Numan, Khan Humaira, A. Awan, S.R. Sohail, Analgesic, anti-inflammatory and antipyretic activities of stem extract of Zizyphus oxyphylla edgew, World J. Zool. 10 (2) (2015) 107–111. [56] W.A. Kaleem, N. Muhammad, M. Qayum, S. Khan, M. Zia-ul-haq, M.I. Choudhary, Biological screening of oils from Zizyphus oxyphylla edgew, Pak. J. Bot. 44 (6) (2012) 1973–1976. [57] P.H.M. Nunes, L.C. Marinho, M.L.R.L. Nunes, E.O. Soares, Antipyretic activity of an aqueous extract of Zizyphus joazeiro Mart (Rhamnaceae), Braz. J. Med. Biol. Res. 20 (1987) 599–601.

[58] M. Nisar, W.A. Kaleem, M. Qayum, I.K. Marwat, M. Zia-ul-Haq, I. Ali, M.I. Choudhary, Biological screening of Ziziphus oxyphylla Edgew stem, Pak. J. Bot. 43 (2011) 311–317. [59] V. Jakus, H. Hrnciarova, J. Carsky, B. Krahulec, N. Rietbrock, Inhibition of nonenzymativ protein glycation and lipid peroxidation by drugs with antioxidant activity, Life Sci. 65 (1999) 1991–1993. [60] R.P. Dearlove, P. Greenspan, D.K. Hartle, R.B. Swanson, J.L. Hargrove, Inhibition of protein glycation by extracts of culinary herb and spices, J. Med. Food 1 (2008) 275–281. [61] M. Hamayun, Ethnobotany of Some Useful Trees of Hindu-Kush Mountain Region: A Case Study of Swat Kohistan, District Swat Pakistan, (2001–2004) . [62] M. Qayum, M. Nisar, M.R. Shah, M. Zia-ul-Haq, W.A. Kaleem, I.K. Marwat, _ Biological screening of oils from Impatiens bicolor Royle, Pak. J. Bot. 441 (2012) 259–355. [63] A. Ceriello, D. Giugliano, A. Quatraro, C. Donzella, G. Dipalo, P.J. Lefebvre, Vitamin E reduction of protein glycosylation in diabetes. New prospect for prevention of diabetic complications? Diabetes Care 14 (1991) 68–72. [64] Y. Duraisamy, J. Gaffney, M. Slevin, C.A. Smith, K. Williamson, N. Ahmed, Aminosalicylic acid reduces the antiproliferative effect of hyperglycaemia, advanced glycation endproducts and glycated basic fibroblast growth factor in cultured bovine. aortic endothelial cells: comparison with aminoguanidine, Mol. Cell. Biochem. 246 (2003) 143–153 s.n..