Ethnomedicinal Survey and a Quantitative Analysis of Bioactive Plants Used by Sonowal Kachari Community of Assam

Background In recent times, cases of infectious and malignant diseases are increasing in an exponential manner. The treatments to these diseases face drawbacks of specificity and high costs. Due to this, many patients seek alternative or complementary methods of treatment. Ethnomedicine is the first and foremost choice in this regard. However due to cultural belief no proper documentation with scientific validation is available. The present study intended to validate the potentiality of the ethnomedicinal plants of Sonowal Kachari community of Assam, quantitatively and qualitatively along with a few bioactivity studies of oxidative stress and antioxidant defences in a murine immune model. Methods The study was based on extensive ethnomedicinal field survey for 1 year period from 2015-2016. Data was analysed and most potent plant was short listed using disease consensus index (DCI), phytochemical screening and 2,2-diphenylpicrylhydrazyl (DPPH) assay. An effective dose was determined by calculating IC50 with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction (MTT) assay in murine splenic macrophages. Bioactivity were studied in lipopolysaccharide (LPS) stimulated murine splenic macrophages for oxidative stress and antioxidant parameters.


Introduction
With recent advancement of technologies, countries with strong health care systems have the advantage of early diagnosis and advanced treatment of life-threatening diseases like cancer leading to better survival rates among the patients (1). However, cancer still remains the second leading cause of death around the world, resulting in nine million diagnosed cases and around five million deaths each year (2). Till date available treatment of cancers suffers from drawbacks of toxicity and non-specificity. Due to various drawbacks and high costs of therapy, many cancer patients seek alternative and/or complementary methods of treatment. Ethnomedicine is the first and foremost choice in this regard. Natural products account for more than half of all modern medications in clinical use. Many plant derivatives have been employed with variable levels of effectiveness and to provide a better treatment for various cancers. A wide range of plant extracts have been shown to have anticancer and/or anticarcinogenic properties. Currently, the chemotherapeutic treatment of tumors involves a number of plant-based compounds that are either in use or in clinical studies. Plant-based ethnobotanical leads are among the most effective agents in the hunt for anticancer drugs. There is a growing interest to study the natural drugs which claim to have anti-inflammatory and anticancer effects. Moreover, if such natural drugs are found to possess immunomodulatory properties, this could act as a double-edged sword employing both chemotherapy and immune targeting of cancer cells. Sonowal Kachari, one of the largest plain tribes of Assam, is confined to the remote areas of Brahmaputra Valley. The ethno-botanical lore of this tribe is very rich. Knowledge on effective medicines acquired by them through experience is usually passed on orally as a guarded secret of certain families (3) and due to this reason ethno-medicinal plants used by this community have remained unexplored and unreported. A few field surveys have been carried out (4) covering documentation of several treatments for different ailments except cancer. Moreover, there are no reports on qualitative and quantitative bioactivity studies related to the documented ethnomedicines of this community so far. Therefore, in the present investigation, we intended to validate the potentiality of the ethnomedicinal plants of Sonowal Kachari community for the bioactive activities by using quantitative tools and test the selected medicinal plant for such bioactivity studies.

2.2.
Ethics approval Mice were maintained, treated and sacrificed in accordance with the guidelines set by the Institutional Ethics Committee (IEC), Assam University, Silchar, India under the approval number of IEC/AUS/2015-027 dt.4/9/15

Area of study
The field survey was carried out in Sonowal Kachari inhabited areas, mostly remote border areas of Dibrugarh, Tinsukia, Dhemaji, Lakhimpur and Jorhat districts of Assam.

Collection of data
The field survey was carried out from 2015 to 2016 for a period of one year and data on plants used to treat tumor were collected through semi-structured questionnaires (5). The traditional medicine practitioners were selected by using snowball technique. The questions were divided into two groups, of which one was for general information about the informant like age, sex, education etc. and the other was for quantitative evaluation. The plant samples were collected from different areas as mentioned in the section entitled as area of study and initially identified using various literature viz. Flora of Assam (6) Flora of British India (7). For further verification, herbarium specimen was deposited in Botanical Survey of India, North Eastern Circle, Shillong.

Quantitative screening of plants based on disease consensus index (DCI)
Data was collected from the informants and evaluated quantitatively using disease consensus index (DCI). This index was used to select highly used plant specimens used to treat specific chronic disease in a specific community (5). It is a comparison based on mathematical aspect (limit theory), the ideal answers of informant reports (Cc) and the ideal answers for each species (Vx).  Where, "x" is any species, "mVxi" is the sum of the individual values obtained for one species within the community; it evaluates knowledge and mentions. "mVx" is the statistical mean of the individual values for one species; it evaluates knowledge. "Cc" is the correlation coefficient, defined as the maximal number of informants who refer to a species; it evaluates mentions. "Pm -0.1 " is the compensation factor, and analyses the dispersion for one species, considering the mode of preparation and parts used.

Preparation of crude plant extracts using Soxhlet method
Five plants with highest quantitative scoring i.e., DCI, were selected for the further study. Different plant parts were collected as per the recommendation of traditional healers of the community used to treat tumors, and then shade dried and powdered. The crude extracts of each sample were prepared using Soxhlet method with petroleum ether (PE), ethyl acetate (EA), acetone (Ac) and methanol (Me). The extracts were then dried using desiccator.

Qualitative Phytochemical Screening
Standard qualitative procedures of Trease and Evans (8), Sofowora (9) and Harbone (10) were followed to prepare PE, EA, Ac and Me extracts from each selected plants and were tested to estimate the presence of important bioactive compounds, mainly secondary metabolites.

Measurement of antioxidant activity by 2,2-diphenylpicrylhydrazyl (DPPH) assay
A slightly modified protocol of (11) was used to determine in vitro antioxidant activity of the selected plant extracts. The dried plant extracts were dissolved in methanol to prepare different concentration of solutions. DPPH methanol solution (6X10 -5 mol/L) was added to the plant extracts and incubated for 30 mins. Methanolic solution of DPPH was taken as control. Absorbance was taken at 515 nm. The antioxidant activity was calculated by the following formula The antioxidant potential was expressed as IC 50 value and plant extract with highest antioxidant potential i.e., lowest IC50 value was selected for bioactivity studies.

Experimental animals
For in-vitro bioactivity studies, 6 to 8 weeks old male Swiss albino mice, body weight of 20±2g were purchased from Pasteur Institute, Shillong, India. Animals were kept in the departmental animal facility under the guidelines of Institutional Ethical Committee (IEC) (Assam University, Silchar, Assam).

Isolation of Splenic macrophage
Splenic macrophages were isolated through density gradient centrifugation at 1500 rpm for 30 mins using Histopaque 1077 (Sigma, USA). The cells were then allowed to adhere to the plastic surface of the Petri dish for 1 hour in 37°C, 5% CO 2 incubator. The non-adherent cells were removed and adherent cell were washed once and re-suspended in culture media at a density of 10 6 cells/ml (12) and further used in biochemical assays.

Dose standardization by MTT assay
Isolated splenic macrophages were divided into 11 experimental groups with 3 replicates (10 6 cells/ml per replicate). Ten (10) doses of selected plant extracts were prepared with the lowest dose of 1µg/ml to the highest dose of 100µg/ml. One untreated group was taken as control. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) reduction assay was use for the determination of cell viability (13) and IC50 (inhibitory concentration for 50% viability) value was calculated to find an effective dose for further experiments.

Biochemical assays
Effect of the selected plant extract on lipopolysaccharide (LPS) induced oxidative stress and its antioxidant efficacy was studied using a dose lower than the IC50 value for three different groups viz. control (C), LPS treated (LT) and LPS with plant extract treated (LPET). Following the protocol of Marklund and Marklund (14), superoxide anion (o 2 -) release was estimated. Estimation of antioxidative enzymes like Superoxide dismutase (SOD), Catalase (CAT) and Glutathione (GSH) was done following the methods of McCord (15), Bergmeyer (16) and Ellman (17) respectively.

Flow chart representation of the experiment
Our entire experimental process is depicted in a precise manner with the help of flow diagram ( Figure 5)

Statistical analysis
The quantitative phytochemical screening of selected plants was done by one plant value i.e., a kind of binary evaluation-"1" or "yes" for knowledge and "0" or "no" for lack of such knowledge about the questions asked. Qualitative phytochemical screening, in vitro antioxidative activity were measured in triplicates.
All in vitro bioactivity studies were carried out in triplicates. The values were expressed as mean ± standard error mean (SEM) and analyzed with non-linear regression Student's t test and one way ANOVA. Significance were marked at p<0.05 and the significance level has been mentioned in the respective graphs. Statistical calculations were performed in SPSS v20 and data representations were done in the GraphPad Prism 8 software

3.1.
Collection of data and quantitative screening based on disease consensus index (DCI) Types of plants used by the Sonowal community for treating tumors are quite less in number. Therefore, plants used in other chronic pathological conditions like piles, boils, wounds which if left untreated might leads to the development of cancer were also considered in our study. In this field survey 45 types of plants were reported by 31 informants. The mode of application of the plant parts along with its scientific name followed vernacular name and DCI were tabulated in Table 1. DCI was used to evaluate collected data quantitatively and five plants with highest DCI were selected for further studies. We found that Oroxyllum indicum has highest DCI value of 0.611 followed by Abelmoschus moschatus 0.355, Zanthozylum nitidum 0.341, Eupatorium odoratum 0.311 and Callicarpa arborea 0.292.

3.2.
Qualitative phytochemical screening The extracts of A. moschatus, C. arborea, E. odoratum, O. indicum and X. nitidum were prepared using the prescribed plant parts with PE, EA, Ac and Me (Table 2) and employed for qualitative screening. From qualitative phytochemical screening it was found that the methanol extracts of A. moschatus (AMM), C. arborea (CAM), O. indicum (OIM), Z. nitidum (ZNM) and ethyl acetate extract of Z. nitidum (ZNE) showed the presence of maximum no. of phytochemicals (Table 3). Therefore, all five selected extracts were subjected for further analysis.

3.3.
DPPH assay DPPH is the stable free radical used to test the free radicle scavenging activity and to evaluate antioxidant potential of plant extract. In our studies OIM extract showed highest antioxidant potential with IC 50 of 0.074 mg/ml followed by ZNE 0.348 mg/ml, AMM 0.591 mg/ml, CAM 0.615 mg/ml and ZNM 0.619 mg/ml (Figure 4). Therefore, OIM extract was selected for bioactivity study in murine splenic macrophages.

3.4.
Standardization of dose MTT is a yellow-coloured water soluble tetrazolium salt which can be reduced by viable cells with active metabolism into purple insoluble formazan, i.e., colour formation is inversely proportional to the cytotoxicity. In our experiment we found the IC 50 of OIM is 47.64 µg/ml ( Figure 5). From the reports by US NCI plant screening program, a crude plant extract can exhibit in vitro cytotoxicity effect with an IC 50 ≤ 20µg/ml (18). Therefore, OIM with higher IC 50 value is less toxic to the normal splenic macrophages in our experimental studies. Hence, we take a dose of 4.76 µg/ml for our further studies.

3.5.
Effects of OIM extract on in vitro oxidative stress The LPS induced oxidative stress in LT group (0.0756±0.002) results in rapid increase of O 2 anion release as compared to control (0.0378±0.002), however the LPET group (0.0596±0.001) showed reduction in superoxide anion release after OIM extract treatment ( Figure 6A). The exposure of LPS lower down the release antioxidant enzyme SOD ( Figure 6B), however plant extract enhances its production in the LPET group. The similar pattern was seen in cases of other antioxidat enzymes viz. CAT ( Figure 6C) and GSH ( Figure 6D).

Discussion
Malignant tumors are one of the major causes of death in present century. Despite of having a number of treatments for the cause, due to the side effects of used drugs especially chemotherapeutic drugs, non-specificity and high-cost people seek alternative therapy. In this regard plant-based medicines are the first and foremost choice. Many plant-based medicines have been clinically tested and incorporated in modern medical system. The source of these phytomedicines are the ethnomedicines of various tribes, as these have been screened and qualified by the experiences of many generations for their easy accessibility, efficiency with lower side effects. Only drawback of these ethnomedicinal system is that they are not documented properly and passed to next generation "orally". Sonowal Kachari tribe of Assam is a native tribe with rich ethnobotanical lore and have same drawback as other tribes on their ethnomedicinal practices. No. of field survey for the documentation of traditional knowledge of this tribe are few in numbers (4), (19), (20), however their qualitative and quantitative analysis was not done yet. Therefore, the present study is designed to document the ethnomedicinal knowledge of this tribe qualitatively, quantitatively and estimate their bioactivity in murine splenic macrophages.
During the survey it was found that Sonowal Kacharis' have some supernatural belief regarding some persistent disease like cancer, tuberculosis etc. Therefore, they do not treat this disease directly. As cancer has multistage developmental form, pre-cancerous lesions to malignant tumor stage (21), therefore data for treatment of chronic pre-cancerous lesions, cuts and wounds etc. have also been collected for the experimental purpose.
Secondary metabolites of plants are the main contributor for bioactivity. These metabolites act as an antioxidant, immunomodulator, antimicrobial, anticancer and anti-inflammatory agents in various pathological illness. Present study of plants with highest quantitative value reveals the presence of different secondary metabolites. Among 20 different extracts OIM, AMM, ZNA, ZNM and CAM found to have highest number of phytochemicals including flavonoids, alkaloids, tannins etc. and may be responsible for their antioxidative efficacy. The instantaneous and effective way for the assessment of antioxidant activity of bioactive compounds is to study DPPH free radical scavenging activity. The presence of phenolic components such as flavonoids, polyphenolic acids etc. increases the antioxidative activity, which is expressed as IC 50 . Lower the IC 50 value, higher the antioxidative activity. In this study, methanol leaves extract of OIM showed highest antioxidative activity among other different tested extracts. Prolong oxidative stress leads to chronic inflammation, which causes many chronic diseases like cancer, diabetes, cardiovascular disease etc. Due to oxidative stress extensive amount of free radicals and reactive oxygen species (ROS) are produced in the cell leading to cell death. Antioxidant plays an important role in reducing the oxidative stress. The cell has developed various enzymatic (SOD, CAT and GPx) and non-enzymatic (vitamin C and E, GSH etc.) antioxidants to scavenge oxidative stress (31). The antioxidative efficacy of a compound must be tested in a biological system to validate biochemical results. In the present study antioxidative efficacy of OIM was tested in a LPS stimulated immunecompromise model of murine splenic macrophages. LPS a cellular component of gram negative bacteria is a classic inflammasome activator (32). In recent study, of Halawa et. al (2013) (33) they reported the oxidative stress and apoptosis inducing properties of LPS. It also been reported for promoting metastasis in cancer cells (34), (32). In this study, similar result with Halawa et. al (2013) (33) was observed for LPS treated group i.e. release of high amount of superoxide anion (O 2 -), which was reduced after OIM treatment and its effect on the cell number was observed under the microscope (Figure 7). This shows that OIM can reduce oxidative stress. The elevated level of antioxidative enzymes SOD, CAT and GSH indicates the activation of the antioxidant defence system in the immune-compromised cells by OIM. Hence, supporting the results of the phytochemical analysis and DPPH assay.

Conclusion:
Thus, from the study it is found that Sonowal Kachari tribe is rich in its ethnomedicinal knowledge and protective about their cultural belief. The medicinal plants used by the community have high amounts of phytochemicals that believe to enhance their medicinal efficacy. Systemic pharmacological trial for antioxidative enzymes with highest phytochemical containing extract i.e., OIM validate estimated biochemical results. Further systemic analysis is needed to validate the indigenous use of medicinal plants and incorporation of the traditional knowledge in modern healthcare system. This will add newer frontiers to medicinal research. Moreover, such type of ethnomedicinal studies help in the preservation of undocumented knowledge of indigenous tribes.