A review on ethnopharmacological utility, traditional knowledge and phytochemistry of Aristolochia species in Assam, India

Aristolochia L. (Aristolochiaceae) is widely used throughout South-East Asia for the treatment of several diseases. Different species of this genus are known by similar local names in Assam. This review aims to provide up-to-date information on Aristolochia species distributed in Assam, including its traditional uses, phytochemical and pharmacological properties, in exploring future therapeutic and scientific potentials. The information on ethnobotany, phytochemistry and pharmacological aspects were collected by performing literature searches. Assam hosts a total of six species of Aristolochia. The taxonomy and distribution are presented. Traditionally the tubers are used by the local people to treat stomach pain, malaria, dysentery, high blood pressure, body pain, urinary tract infections, headache, impotency etc. It has considerable pharmacological properties including antimicrobial, antioxidant, anti-inflammatory, anti-cancer, anti-diabetic, anti-fertility, anti-venom, anti-diarrhoeal, anti-pruritic, anti-feedant and toxicological activities. Approximately a total of 200 compounds have been isolated from these species. So far, pharmacological investigations are only done on three Aristolochia species, whereas the other three are simultaneously used for the same purposes. Most of the medicinal properties attributed to these Aristolochia, have not yet been investigated and proven under a scientific study. This highlights the importance of Aristolochia as a valuable candidate for future studies.


Introduction Introduction Introduction Introduction
Medicinal plants serve humans as a great source of therapeutics and pharmaceutical manufacturing. The practices of using medicinal plants in the treatment of common diseases are part of the traditional knowledge among the different communities throughout the world. The dependencies of the traditional communities on the naturally occurring herbs are due to better cultural acceptability, compatibility and adaptability of the  Aristolochia spp. has been used in traditional medicine by different communities around its occurrence for a long time. The tubers of Aristolochia are used against a multitude of ailments in Assam. Correlating the ethnomedicinal reports with modern pharmacological and phytochemistry studies, shows consistency with the latest findings. A. acuminata is used to treat diarrhoea and dysentery by several tribes residing in Assam (Rao, 2019). A. saccata is used to treat stomach ache, constipation, dysentery, fever, body pain, jaundice, sprains and fracture by the Karbi, Tiwa, Pnar and Bodo-Kachari people residing in Karbi-Anglong districts of Assam (Basumatary et al., 2014;Teron, 2019). The population in Majuli Island and around Gibbon Wildlife Sanctuary uses its roots to treat tonsillitis, cough, piles, malaria fever and diarrhoea (Sarmah and Saikia, 2014;. The roots of A. indica are used to heal wounds and to enhance fertility in males by the traditional healers of Dhemaji district of Assam (Taid et al., 2014). It has also been reported to be used by the people of Dibru-Saikhowa Biosphere Reserve for the treatment of certain asthmatic problems and skin diseases such as leucoderma (Nath et al., 2008;Purkayastha et al., 2007). The Deori, Muttak and Nepalese community residing in the Dibrugarh district of Assam reported using the decoction of the leaves of A. indica to treat dysentery, diarrhoea and melena (Borah et al., 2006). A. cathcartii is used against stomach aches, urinogenital disorders and as an insect repellent by the people in and around Manas Biosphere Reserve (Paul et al., 2011 a, b). Similarly, the roots of A. assamica and A. cathcartii are used to treat stomach pain, malaria, dysentery, high blood pressure, body pain, urinary tract infections, headache and cough by the fringe people of Behali reserve forest in Biswanath district (Borah et al., 2020). However, several other ethnomedicinal reports have been published for the studied species of Aristolochia outside Assam are shown in Table 1.  Panda, 2010;Sambandan and Dhatchanamoorthy, 2012;Saradha et al., 2017;Sen, 2008;Senthilkumar et al., 2006;Shivakumar and Parashurama, 2014;Shivanna and Rajakumar, 2011;Shukla et al., 2010;Silambarasan et al., 2017;Sivaperumal et al., 2009;Sivasankari et al., 2014;Sudeesh et al., 2012;Sulochana et al., 2014;Swarnalatha et al., 2017;Tiwari and Yadav, 2017;Tripathi and Sikarwar, 2013;Usha et al., 2015;Vijayan et al., 2007;Yadav and Prakash, 2014;Yesodharan and Sujana, 2007 A. acuminata  Biswas et al., 2010;Britto and Mahesh, 2007;Dey et al., 2012;Devi Prasad et al., 2013;Ignacimuthu and Ayyanar, 2005;Kayang, 2007;Bose et al., 2014;Rajashekharan et al., 1989;Rahman et al., 2007;Reang et al., 2016;Sulochana et al., 2014;Silambarasan et al.,  The phytochemical analysis is must to justify the scientific accuracy in the usage of herbal medicine and unearth the basis of treating diseases efficiently. During the last couple of decades, extensive studies were done on the phytochemical constituents found in various plant species and the genus Aristolochia was no exception.
Similarly, aerial parts of A. indica were also studied by disc diffusion method against Pseudomonas aeruginosa, Basillus subtilis, Staphylococcus aureus, Escherichia coli, Bacillus sphaericus (syn. of Lysinibacillus sphaericus), Salmonella typhimurium. The extracts showed a moderate antibacterial activity (Shafi, 2002). Murugan and Mohan (2012)  The leaf extract caused high inhibition of B. coagulans followed by B. subtilis and the least inhibition caused by leaf extract was recorded against S. aureus. In the case of flower extract, S. typhi and B. coagulans were inhibited to a higher extent when compared to other bacteria. Antifungal activity was studied by poisoned food technique against test fungi namely Bipolaris sorokiniana (from root rot of wheat), Fusarium oxysporum f.sp. zingiberi (from rhizome rot of ginger), Colletotrichum capsici (from anthracnose of chilli) and Curvularia sp. (from mouldy grains of sorghum) and the results revealed that F. oxysporum displayed higher susceptibility to leaf and flower extracts followed by Curvularia sp., B. sorokiniana and C. capsici. Umamaheshwari and Murthy (2012) studied against Bacillus subtilis, five different antibiotics namely Ciprofloxacin, Nitrofurantoin, Ofloxacin, Pefloxacin and Sparfloxacin were used as standard, results showed that the root extracts exhibited different degrees of antibacterial activity of which butanol extract of inhibition zone (2.4 cm) and ether extract (2.0 cm) showed maximum activity.

Antioxidant activity
The free radical scavenging activity of methanolic root extracts of A. acuminata was tested by Hadem et al. (2016) by DPPH method and, found significant value as compared to standard compound ascorbic acid. At 1000 μg/ml concentration, aqueous stem extract of A. indica showed higher scavenging activity of 66.66±4.67% compared to chloroform leaf extract of 48.33±3.38% in DPPH method (Subramaniyan et al., 2015). Naik et al. (2015) found the ethyl alcohol extracts of leaves and flowers of A. indica at 100µg/ml concentration showed 48.68% and 10.52% DPPH radical scavenging activity respectively where ascorbic acid was used as standard. The aerial parts of A. indica exhibited IC50 value of 7.325 µg/ml at 25 µg/ml concentration when tested by DPPH radical scavenging method using ascorbic acid as standard and IC50 value of 8.498 µg/ml at 10 µg/ml concentration when tested by superoxide anion radical scavenging method with curcumin as standard (Karan et al., 2012). Thirugnanasampandan et al. (2008) studied the antioxidant activities of both A. acuminata and A. indica using three solvents e.g., petroleum ether, chloroform and ethyl acetate extract. Among the extracts, the highest reducing power activity has shown by the ethyl acetate extract of A. acuminata (1.28%) and A. indica (1.01%). In Ammonium thiocyanate assay, petroleum ether (10 ml) extract of A. acuminata showed the highest activity of 57.42% and A. indica ethyl acetate extract showed the highest activity of 40.21% compared to Linoleic acid.
Anti-inflammatory activity Ethyl acetate and ethanol extracts of A. acuminata roots at doses 200 and 400 mg/kg produced a significant reduction in the Carrageenan-induced paw edema on Wistar albino rats. The test samples exhibited an inhibitory effect for both COX and LOX enzymes, in in-vitro MTT colorimetric assay. Among the isolated phytoconstituents from the plant "Kaempferol" was responsible for the highest inhibition of PGE2 and LTB4 at 87.7% and 91.4% released from calcium ionophore and LPS IFN γ-stimulated macrophages than standard drug indomethacin (Battu et al., 2011). Aristolactam I and (-) Hinokinin isolated from A. indica also exerted anti-inflammatory effects and inhibited the production of IL-6 and cytokines TNF-α in LPS-stimulated THP-1 cells (Desai et al., 2014). Retardation of inflammation has resulted when combined administration of A. indica plant extract and venom Ichthyocrinotoxin administered on Carrageenan induced male albino rats (Das et al., 2010). Ethanolic extract of A. indica roots at dose 150 mg/kg showed a potent anti-inflammatory effect on compound 48/80 induced paw edema in Wistar male albino rats (Mathew et al., 2011).

Anti-cancer activity
Hepatocellular carcinoma (HCC) in Swiss albino BALB/c mice was induced by carcinogen diethylnitrosamine (DEN) which elevates aspartate transaminases, alanine transaminase, alkaline phosphatase activities. A. acuminata root extract significantly attenuated the increased activities of these marker enzymes (Hadem et al., 2014). It was found the root extracts had lowered the levels of tumour necrosis factor-α (TNF-α) levels and nuclear factor kappa-B (NF-κB) activation when analysed the serum and nuclear extracts of DEN induced hepatocellular carcinoma in Albino BALB/c mice. Leaves and stem extracts of this plant also showed chemo-preventive potentiality when tested against six human cancer cell lines (Garg et al., 2007). Fractions of A. acuminata root aqueous-methanol extract of 2.5-5mg/ml concentration exhibited the highest inhibition with IC50 value of 0.320 mg/ml and induced the effective apoptotic activity determined by MTT assay in HeLa cells (Hadem et al., 2019).
The chloroform leaves extract of A. indica showed an inhibitory effect at IC50 value at 347 µg/ml compared to the standard anti-cancer therapy drug Taxol when evaluated in human breast cancer cell line (MCF-7 Michigan Cancer Foundation-7) by MTT assay (Subramaniyan et al., 2015). alloxan induced diabetic mellitus in Sprague Dawley rats and compared with the oral hypoglycemic agent glibenclamide (10 mg/kg). The effect of crude extract on blood glucose levels was measured at various time intervals of 0, 1, 2, 4, 6 and 8 hours. The dose of 400 mg/kg of the crude extract produced a significant maximum fall of 28.94 ± 2.8 on the blood glucose levels of diabetic rats after 6 hours of the treatment compared with disease control group (Goverdhan et al., 2008).

Anti-fertility activity
The anti-fertility activity was evaluated by determining the anti-implantation and early abortifacient activity of ethanolic extract of A. acuminata leaves in Wistar rats of either sex orally at the doses of 100 and 200 mg/kg considering 1% Tween 80 as control drug showed significant (100%) antifertility activity on 200 mg/kg in female rats by a significant reduction in the number of corpora lutea and increase in the number of resorptions (Balaji et al., 2004). The post-coital administration of A. indica ethanolic root extract decreased fertility in both Wistar rats and hamsters (Che et al., 1984).

Anti-pruritic activity
Compound 48/80 induced scratched behaviour model was used to evaluate the scratching response of A. indica root. The ethanolic plant extract at the dose of 150 mg/kg showed significant effect and decreased the scratching incidence (Mathew et al., 2011). The wound healing potency A. saccata leaf extract was studied by Bolla et al. (2019). In vitro cell-based scratch assay in L929 cells resulted after 48 hours of treatment with 125 μg/mL of plant extract closed the gap created by the scratch by 93.525%. The extracellular matrix (ECM) factor, collagen type-1 might be enhanced by the plant extract which initiated the migration of fibroblasts (Bolla et al., 2019).
Anti-feedant activity Antifeedant activity studied by Baskar et al. (2011) reported the leaf extract of A. acuminata was more toxic than the root extract. Maximum anti-feedant activities of 56.06% and 49.86% were recorded on ethyl acetate and hexane leaf extracts of A. acuminata at 5.0% concentration against Spodoptera litura using leaf disc no-choice method while the root ethyl acetate extract expressed minimum activity of 31.71%. At the same concentration, the ethyl acetate leaf extract exhibited the highest larvicidal activity (40.66%) and pupicidal activity (68.06%). Significant larval toxicity showed by A. indica leaf against Anopheles stephensi. The formulation of Aristolochic acid I at concentrations of 1000 ppm reduced the survival of all larval instars (Murugan et al., 2015;Pradeepa et al., 2015).

Toxicology
The aristolochic acids found among the species of Aristolochia are famous for nephrotoxicity after the tragic Belgian cohort where the women have taken the weight reducing pills contained Chinese herb, Stephania tetrandra was inadvertently replaced by aristolochic acid-containing A. fangchi were reported to suffering renal interstitial fibrosis (Balachandran et al., 2005;Debelle et al., 2008). The nephrotoxic and carcinogenic properties of the compound aristolochic acids have been recognised and can cause permanent kidney injury, renal failure (Han et al., 2019). The toxicological risk on the consumption of drugs made up of A. indica depends upon several factors like processing, preparation of drugs and mode of administration (Michl et al., 2013). In the quality control of the herbal recipe Homnawakod, Tripatara et al. (2012) demonstrated that one of its formulations e.g., the dried roots of A. acuminata were not causing nephrotoxicity in rats even the daily administration for 21 days. The acute toxicity study revealed no cytotoxic effects of A. acuminata leaves and root, A. indica aerial parts and roots when tested in both Swiss albino mice and Wistar albino rats (Balaji et al., 2004;Battu et al., 2011;Mathew et al., 2011;Karan et al., 2012). Leaves of A. saccata exhibited mild toxicity against L929 fibroblast cell line at minimum percentage resulted in the death of only 2.88% of cells (Bolla et al., 2019). Michl et al. (2013) also reported the contents of Aristolochic acid is higher in leaves, fruits and young stem than roots and woody stems. Mostly the roots of the Aristolochia sp. are used for a different form of traditional medicine, which arise a problem, as most plants are uprooted directly from the wild before reaching reproductive maturity. This poses a serious threat and is also elucidated by Kayang (2007). However, effective planning on cultivation and management of Aristolochia on a small scale can help address this issue, as well as introduction in the home gardens can solve this problem. They can also be planted as beautiful ornamental. The attractive flowers add aesthetic value to its present traditional utilities.

Conclusions Conclusions Conclusions Conclusions
The plants of the genus Aristolochia have always been recognized as plants of high medicinal importance by the people of Northeast India. But in recent years, the genus Aristolochia L. has been recognized globally for possessing remarkable medicinal value and is reportedly used by people throughout the Indian sub-continent against various diseases and illnesses such as snake bites, muscular ailments, lung, liver and gastrointestinal disorders etc. Hence, in this present study, we have comprehensively reviewed the traditional knowledge on six species of Aristolochia found in the northeastern state of Assam along with the various phytoconstituents present in those species. Also, the various properties viz. antimicrobial, antioxidant, anti-inflammatory, anticancer, anti-diabetic, anti-fertility, anti-venom, anti-diarrhoeal, anti-pruritic, antifeedant and toxic activity exhibited by various parts of the plant. In short, this review is designed to provide insight into the necessity of further research of important plant compounds to investigate and develop new drugs. Additionally, more comprehensive reviews regarding the activity of the compounds found in Aristolochia will help in further development of using Aristolochia as an effective drug. Hopefully, these studies will explore the full potential of Aristolochia and optimize its use as a promising herbal medicine, thereby promoting global health.
All authors read and approved the final manuscript.