Phytochemical analysis and bioactivity reports of ethnomedicinal plants from West Bengal, India

Abstract The biodiversity-rich forests of the Jhargram subdivision of West Bengal, India houses many lesser-known prospective plants. Four ethnomedicinal plants from this locality—Cleistanthus collinus, Tiliacora racemosa, Eupatorium odoratum, and Sida acuta reported for traditional medical uses by local forest tribes have been analyzed for phytochemical constituents and bioactivity potential, viz., antioxidant, antibacterial and antitumor activity. Cleistanthus and Tiliacora plants were rich in alkaloids while Eupatorium and Sida showed tannin abundance. Tiliacora showed maximum alkaloid content, that is, 711 mg strychnine equivalent/gm dry weight. Consequently, these plant extracts showed decent antioxidant activity which is reflected in their antibacterial and antitumor potencies. Cleistanthus showed strong bactericidal activity against Gram-negative bacteria, particularly against Klebsiella pneumoniae and Pseudomonas aeruginosa, while Tiliacora showed robust antitumor activity against cervical cancer cells SiHa at a 50% inhibitory concentration (IC50) of 86 µg/ml. Hence, the biodiversity-rich Jhargram forest should be conserved to protect the potential repertoire for ethnomedicinal plants. Graphical Abstract


Introduction
The Indian subcontinent is one of the richest abodes of medicinal plants building the cornerstone of the oldest medicinal system-Ayurveda. According to World Health Organization (WHO 2019), 88% global population relies on phytotherapeutic approaches. The most noteworthy fact about Indian forests is their traditional practice by indigenous communities. In this context, West Bengal, particularly the 'Paschim Medinipur' district's Jhargram region contains the Amlachati Medicinal Plant Garden with the largest medicinal plants collection in the country (Goswami and Das 2018). However, no studies till date have reported their bioactivity. Since this region is inhabited by tribal communities like Santhals, Mundas, Lodhas, Bhumijs, Oraon, and Kherias, their ancient practice of using plant parts as medicines can be maneuvered into the modern-day medicine formulations. Hence, this research aimed to study the most abundant ethnomedicinal shrubs of Jhargram namely, Cleistanthus collinus, Tiliacora racemosa, Eupatorium odoratum, and Sida acuta.
Alcoholic extracts from the plant specimen C. collinus [(Roxb.) Benth.] from the Euphorbiaceae family has been used in gastrointestinal disorders, cancer and fungal diseases. The chemical composition of Cleistanthus reveals the presence of lignans, terpenoids like lupeol, cleistanthol, and glycosides cleistanthin A and B (Fan et al. 2020). Eupatorium odoratum (L.) belonging to the Asteraceae family is a common shrub and can be used as an immunomodulator, hepatoprotective, antidiabetic, antihypertensive, and so on (Umukoro and Ashorobi2006). These wide spectrum bioactivities are owed to terpenoids (eupatoric acid), flavonoids (isokuranetin), essential oils (a-pinene, camphor, b-caryophyllene), and so on (Suksamrarn et al. 2004;Joshi 2013). Tiliacora racemosa Colebr. from the Menispermaceae family is mentioned as Krishnavetra in Ayurveda, particularly for treating cancer. It is the biggest storehouse of bisbenzylisoquinoline (BBI) alkaloids, chiefly tiliacorine and tiliacorinine (Chakraborty et al. 2004;Pal et al. 2021). The fourth specimen, Sida acuta from Malvaceae has various therapeutic uses owing to indoloquinoline alkaloids (cryptolepine) along with steroidal (b-sitasterol and stigmasterol) and phenolic compounds (evofolin A/B) (Karou et al. 2007).
This study is aimed to provide an exemplary overview of the natural repository of the Jhargram subdivision of West Bengal, India. The present investigation is a phytochemical composition analysis and bioactivity report of four representative ethnomedicinal plants, to serve as a pilot study for future drug development.

Phytochemical composition of the plant extracts
The methanol and ethyl acetate extracts stand superior to hexane and aqueous extracts in terms of phytochemical extraction, proving the plant specimens to be loaded with polar metabolites. In the case of Cleistanthus collinus and Eupatorium odoratum, alkaloid content seemed to be higher (308.8 ± 15.3 mg SE (strychnine equivalent)/gm DW (dry weight) and 383.8 ± 15.4 mg SE/gm DW respectively) followed by tannin and phenol content ( Figure S1A and S1B), while in case of Tiliacora racemosa, the alkaloid content is maximum (711.38 ± 8.2 mg SE/gm DW) ( Figure S1C). Lastly, for Sida acuta, along with the alkaloid content (324.9 ± 6.28 mg SE/gm DW), tannin content (216.87 ± 5.8 mg CE (catechin equivalent)/gm DW) was also higher ( Figure  S1D). The heatmap contour plot in Figure S1E supports the same notion that methanol and ethyl acetate extracts harbored maximum pharmacological metabolites, among which alkaloid content topped the list, most strikingly in Tiliacora methanol extract ( Figure S1E). This alkaloid content can be owed to the BBI alkaloids, chiefly tiliacorinine as reported by our group previously (Pal et al. 2021). Additionally, the Principal component analysis (PCA) in Figure S1F (for methanol extract) and Figure  S1G (for ethyl acetate extract), show that Eupatorium (Asteraceae) and Tiliacora (Menispermaceae) align closer than Cleistanthus (Euphorbiaceae) and Sida (Malvaceae) which also show a closer association. Table S3 lists the pharmacological metabolite contents. This rich phytochemical composition was reflected in their bioactivity studies: antioxidant activity, antibacterial and antitumor properties.

Rich antioxidant profile of plant extracts
All the plant extracts were evaluated for their antioxidant property as shown in Figures S2A-C and S3. Methanol and ethyl acetate extracts proved consistently superior in harboring antioxidant properties, therefore, Figure S2A only represents methanol and ethyl acetate extracts. Figure S2A shows Cleistanthus methanol extract harbored significant DPPH and H 2 O 2 scavenging effect (EC 50 at 152.39 mg/ml and 141.67 mg/ml) while its ethyl acetate extract showed better metal chelation power (EC 50 at 118.86 mg/ml) and NO scavenging effect (EC 50 at 178.11 mg/ml). On the other hand, Eupatorium ethyl acetate extract showed the best NO scavenging effect (EC 50 at 68.12 mg/ml). Next, Tiliacora methanol extract showed the best DPPH scavenging effect (EC 50 at 94.42 mg/ml). Even, Sida methanol and ethyl acetate extracts did not show significant radical scavenging effect. For FRAP assay ( Figure S2B), Cleistanthus, Eupatorium, and Tiliacora methanol extracts-all showed significantly high FRAP value. Lastly, for the total antioxidant assay ( Figure S2C), Tiliacora methanol extract surpassed all with the highest total antioxidant capacity (79.93 mg AA (ascorbic acid)/gm DW).

Antimicrobial activity of the plant extracts
As detected through agar diffusion techniques at a fixed dose of 500 mg/well, the plant extracts were more effective against Gram-positive bacteria than Gram-negative bacteria. This might be due to the outer membrane in Gram-negative bacteria which limits the penetrability of antimicrobial agents to their targets in the bacterial cell (Vaara, 1992).
The comparative antimicrobial activities have been depicted in Figure S4 and detailed in Table S3. Since the aqueous extracts did not show any significant phytochemical constituent and antioxidant activity, they were not used for the study of the antimicrobial property. The most significant broad-spectrum antimicrobial activity was shown by ethyl acetate extract of Cleistanthus against both Gram-positive and Gramnegative bacteria, particularly more potent against Klebsiella pneumoniae and Pseudomonas aeruginosa than the clinically used streptomycin and chloramphenicol antibiotics. Although Cleistanthus leaf extracts have been previously reported for bactericidal activity (Elangomathavan 2013), this study depicts the antibacterial potency of Cleistanthus against a broader bacterial spectrum and also proves that ethyl acetate is better in extracting the potent antimicrobial compounds from Cleistanthus leaves. Eupatorium ethyl acetate extract also exhibited broad-spectrum activity with inhibition zones comparable to antibiotics, except for Bacillus subtilis and B. cereus. In Tiliacora, the most promising antibacterial activity was shown by the hexane extract against Salmonella enterica and K. pneumoniae. Lastly, in case of Sida, the methanol extract exhibited the most notable activity against both Gram positive and Gram negative bacteria except for S. enterica and K. pneumoniae. This might be due to the presence of alkaloids in this fraction.

Anticancer activity of the plant extracts
All the plant extracts were evaluated for their cytotoxic ability through the MTT reduction assay in cervical cancer cells SiHa as shown in Figure S5A-E. As evident from Figure S5E, Tiliacora methanol extract outshined all other plant extracts with the lowest IC 50 dose of 86.64 ± 3.11 mg/ml (listed in Table S4). Studies on Tiliacora's anticancer potential have been previously reported by our group (Pal et al. 2021), wherein the plant extract containing BBI alkaloids (chiefly tiliacorinine) inhibited SiHa cells by oxidative stress-induced G2/M phase cell cycle arrest and apoptosis. The same experimental dataset has been used here to provide a comparative overview with other ethnomedicinal plants from the same locality. Beyond Tiliacora, the sequence of cytotoxicity followed the trend Cleistanthus > Eupatorium> Sida methanol extracts with IC 50 at 223.41 ± 12.22 mg/ml, 348.27 ± 27.44 mg/ml and 448.11 ± 37.61 mg/ml, respectively ( Figure S5A, S5C-D). With regard to C. collinus, cleistanthin A and B have been depicted to have cytotoxicity in SiHa cells (Kumar et al. 1998;Pradheepkumar and Shanmugam 1999) and this proves that the methanol and ethyl acetate extract possibly harbors cleistanthin A and/or cleistanthin B along with other metabolites, which might be enhancing their cytotoxicity. Although this speculation needs further studies, altogether, Cleistanthus and Tiliacora turn out to be one of the most potent medicinal plants from this region. The cytotoxic potential of Cleistanthus and Tiliacora methanol extracts is also evident from the eroded cellular and nuclear morphology of SiHa cells as shown in Figure S5F. As compared to the spindle-shaped healthy control cells with intact oval nuclei, these nuclei seem condensed, shrunken and at times damaged, while the cellular morphology show damage signs with cells becoming rounded, membrane ruptured and cytoplasmic contents released. The inconsistency between antibacterial and antitumor potency of the four ethnomedicinal plant specimens might be due to the different modes of action of different phytochemicals.

Conclusion
Hence, out of these four ethnomedicinal plants, Cleistanthus and Tiliacora have emerged as the most potent medicinal plants with rich alkaloid content. These plants with significant tribal medicinal usage should be conserved, i.e., the forests of Jhargram region in West Bengal should be taken into serious consideration. This ethnomedicinally rich region upon further scientific probing is diligently capable of delivering medicinal wonders to the world.