Variation in biosynthesis of an effective anticancer secondary metabolite, mahanine in Murraya koenigii, conditional on soil physicochemistry and weather suitability

Murraya koenigii (MK) leaf being a rich source of bioactive secondary metabolites has received inordinate attention in drug development research. Formation of secondary plant metabolite(s) in medicinal plants depends on several factors and in this study the cause of variation in bioavailability and content of a vital bioactive phytochemical, mahanine in the MK leaves from different geographical locations of varying soil properties and weather parameters was determined. Accordingly, MK leaves and soil samples around the plant base in quintuplicate from each site across five states of India at similar time point were collected. Mahanine content was determined and compared among samples from different regions. The quantitative analysis data comprised that MK-leaves of southern part of India contains highest amount of mahanine, which is 16.9 times higher than that of MK-leaves of north-eastern part of India (which measured as the lowest). The results suggested that pH, conductivity and bacterial populations of the soil samples were positively correlated with mahanine content in the MK-leaves. For examples, the average soil pH of the southern India sites was in basic range (8.8 ± 0.6); whereas that of the north-east India sites was in slightly acidic ranges (6.1 ± 0.5) and mean soil conductivity value for the north east India soils was 78.3 ± 16.3 µS/cm against mean value of 432.4 ± 204.5 µs/cm for south India soils. In conclusion, this study proclaims that higher level of bioactive phytochemical, mahanine in MK leaves depending upon geographical location, weather suitability and soil’s physiochemical and microbial parameters of its cultivation sites.

Murraya koenigii (MK) (L.) Spreng is a medicinal plant of high value which generally grown profusely in the tropical and subtropical region of Asia 1 . Secondary metabolites in MK plant leaf are known to exhibit diverse medicinal properties including anti-cancer, anti-diabetics, anti-arthritics activity 2,3 . The MK plant leaf is very popular and has been used in the Indian cuisine for its characteristic aroma 4 . The chemical profiling of MK leaves suggests that the secondary metabolites in MK leaf are several alkaloids, terpenoids, flavonoids and phenolic 5 . Few isolated pure compounds from the MK leaf have been studied thoroughly for their details signal cross talk mechanism in different disease models including cancer 6 . For example, mahanine, a carbazole alkaloid isolated from MK leaves established its ability to combat against diabetes and the different types of cancer 7,8 . In a recent review 9 , we discussed in details the molecular mechanistic insight of the mahanine in different cancer model. In another study, we established mahanine as potent chemotherapeutic option for different subtypes of breast cancer 2 .
The naturally occurring secondary metabolite of a plant system has been shown to defend the exogenous influences such as weather, microbes, bacteria etc. 10 . The direct role of secondary metabolites on plant growth and maturity is still not clear and needs further thorough research 11,12 , although the contribution of that secondary plant metabolite (SPM) towards pharmaceutical drug development has been well recognized for centuries 13 . The list of examples includes taxol, vincristine, penicillin and vinblastine as leading drugs in the market from natural sources 14 . Several other SPM such as ingenol, curcumin, mebutate, betulinic acid, withaferin-A are currently in clinical trials and likely to emerge as effective chemotherapeutic agents in market 15,16 . The combination of chemistry and biology found in nature, is much easier to understand than the laboratory chemistry; and hence it always gives a decent solution as drug for diseases management 17 . Natural products with anti-tumor activity belong to several structurally diverse groups and hence it is difficult to make their classification in a single group 18 . Mechanism of formation and accumulation of those specific toxic material as secondary metabolites in the plant parts is quite ambiguous. In one of our recent study 2 , which involved isolation procedure of mahanine from MK leaves, we witnessed varied range of biosynthesis (of mahanine) depending upon the place, time of collection of the leaf samples as well as weather conditions of the plant sample collection sites. Recently a group of researcher showed that, the seasonal variation of carbazole alkaloids distribution in MK leaves and thereafter emphasized on the potentiality of the EtOAc extract against the cancer treatment 19 . The present study was carried out to comprehend the matrixes behind the formation of such type of variation of secondary metabolite in the plant parts. The primary objectives of this study were to (i) determine the variation of mahanine in MK leaves collected at single time point from different geographical locations of varying soil and weather parameters and (ii) possible association of soil, microbes and weather parameters with the mahanine biosynthesis in MK leaves of diverse locations.

Methods and materials
Selection of sites and collection of samples. The sites for sampling were selected to represent major Murraya koenigii grown areas and also variation in edaphic and climatic factors of plant growth. Accordingly, MK plants were collected from five different sites from each of 5 states of India namely Assam (North-East), West Bengal (East), Maharashtra (West), Andhra Pradesh (South) and Delhi (North)] depending on the geographical location. The sampling at five states across the different geographical location is shown in Fig. 1. The five different MK plants sample sites in a state were located within a distance of approximately 10 km from each other. The selected (unbiasedly) plants were 4-12 years old from their planting time. The exact location of each plant (collection site) was tracked by GPS and recorded. The different samples were collected by multiple collector in a way to accomplish the collection on a same day (15th Jan, 2019). For each plant sampled, leaves from the top shoots and soil samples from the trunk surrounding at a depth of 0-15 cm were collected and placed in a sterile zip lock bag with proper labeling. For the collection of soil samples an area was demarcated at a radius of 1 cm from the periphery of trunk and the composite soil was collected through sterilized trough from 0 to 15 cm depth and placed in a sterile zip bag. All the soil samples from different sites were collected in the same passion. The samples were brought to the laboratory within 24 h of collection from field. The soil samples were divided into two halves. First half was stored at 4 °C for determination of bacterial populations and the other half was shade dried for 5 days and stored for further analysis to determine different chemical parameters. The soil samples were marked as AP for Andhra Pradesh, MH for Maharashtra, DL for Delhi, AS for Assam and WB for West Bengal respectively as per their site of collection with five replication from 1 to 5.
Weather parameter record collection. The detailed weather reports of the last 1 year (from February, 2018 to January, 2019) of selected sites were downloaded from the authenticated source, www.world weath eronl ine.com site 20 . The different weather parameters such as the total rainfall, average humidity and average temperature were plotted to show the spatial and temporal variations.
Soil microbial analysis. Soil rhizosphere microbiota plays a crucial role in formation of plant secondary metabolites and thereof monitoring plant's immune system 21 . Total bacterial population in dry soil samples were determined by serial dilution pour plate method. Shade dried soil samples were carefully crushed individually in sterile motor and pestle into fine powdered and sieved through 100 µm sieve 22 . 1 g of the each finely sieved sample was taken and the serial dilution was performed from 10 3 to 10 6  Analysis of soil physicochemical parameter. The physicochemical parameters such as pH and electrical conductivity (EC) of the shade-dried soil samples (sieved through a 0.5 mm sieve) were determined in 1:5 soil suspensions (w/v; soil/water) with Eutech digital pH meter and an EC meter respectively 24 . The organic carbon was determined by modified Walkley and black titrimetric method and the available phosphorus concentrations was determined in a UV-Vis Spectrophotometer (Shimadzu 1601, Japan) using the Mo-Sb colourimetric method 24,25 . Total nitrogen for soil samples was estimated by the method of Willits et al. using Kelplus-Classic DXVA automatic nitrogen analysis equipment 24 . Chloride concentrations were determined by titrimetric method 25 .  www.nature.com/scientificreports/ The total moisture content was determined by subtracting the final weight from the compounding initial weight and expressed in percentage.

Reagents.
Methanol and water (HPLC grade), methanol (analytical grade), ethyl acetate, formic acid were purchased from Merck Millipore, USA. Analytical grade pre-coated silica gel chromatography plate was purchased from Merck Millipore, USA. All the chemicals used for the analysis of physicochemical and micronutrients parameters were purchased from Merck, India. The other reagents used in the study, were procured from Sigma Aldrich, USA.

Solvent extraction of leaf and analysis.
Extraction. Pre-weighed (100 g) washed leaves of each sample was separately ground in a mixer grinder with a pre-determined amount of methanol and soaked for 72 h to complete extraction 2 . The methanol extraction procedure was repeated for all the samples to confirm the completion of the extraction. The total methanol extract for each leaf sample were completely dried separately first in rotary evaporator (for the organic solvent) and latter in lyophilizer (for the aqueous part). Further, the methanolic extract for each sample was fractionated separately by EtOAc and water layering (70:30; v/v) and confirm the presence of mahanine in EtOAc fraction only (by spotting the EtOAc and water fraction with standard mahanine in TLC). The EtOAc fraction was dried in a rotary evaporator and stored in a vial at − 20 °C until further use. The yield of ethyl acetate fraction for each leaf sample was calculated.
HPLC analysis. High performance liquid Chromatography (HPLC) analysis was performed to quantify the mahanine content in the EtOAc fraction of the MK leaves of each sample separately. A standard curve was prepared with the standard mahanine isolated and purified in our previous study 2 in a dose range from 0 to 500 µg/ mL. The HPLC method that we have followed to run the standard compound and/or extract samples in this study was described by us previously 2 .
HPTLC analysis. High Performance Thin Layer Chromatography (HPTLC) was performed to understand the signature profiling of the phytochemicals in the MK leaves that were collected from five different zone of India. Single EtOAC fraction was randomly selected from each zone was used for spotting. A total of five EtOAc fractions (one from each zone), with standard mahanine were spotted in a pre-coated silica gel 60 F254 plates. The plates were developed in a solvent system containing chloroform: methanol (95:5) (v/v) + 0.1% formic acid (v/v) and visualized under 254 nm (in a TLC scanner).

Statistical analysis.
For comparison of a parameter between the two places the statistical analysis were performed by unpaired student t-test. All statistical tests were two-sided with 95% confidence interval (CI). A significance level was considered at P < 0.05. Statistical analyses were performed in GraphPad Prism 6.05 software.

Results
Analysis of total moisture content in fresh MK leaves. Moisture content of a plant material affects the secondary metabolite extraction from plant parts 26,27 . As we have extracted the mahanine from the fresh leaves of different MK trees of varying location, it was necessary to check the moisture content of all the collected samples. The total average moisture content of the MK leaves varied from ~ 61 to 64% and did not vary significantly between locations ( Fig. 2A). The moisture content was lowest (60.8 ± 2.9%) in the MK leaves collected from Assam (AS) state and highest (64.1 ± 0.7%) in MK leaves collected from West Bengal (WB) ( Fig. 2A).
Yield of the extract and quantification of mahanine content. The complete methanol extract was separately lyophilized, weighed and the extract yield was determined for the leaves collected from different sites. The yield of methanolic extracts were significantly less in Andhra Pradesh (AP) and Maharashtra (MH) compared to that of Assam (AS) or Delhi (DL) (Fig. 2B). Percentage yield of methanol extract of the MK leaves collected from AP sites were found to be 4.9 ± 0.3%, whereas samples from AS site yielded 7.7 ± 1.6% (Fig. 2B). Further to quantify the yield of EtOAc fraction from methanolic extract, a known quantity of methanol extract was fractionated with EtOAc and water (70:30 v/v). MK leaves samples from MH (15.1 ± 0.9%), WB (14.3 ± 2.6%), DL (13.5 ± 7.4%) and AS (14.2 ± 4.0) yielded the highest amount of EtOAc fraction whereas AP samples yielded lowest amount (13.0 ± 1.6%) of EtOAc fraction per unit quantity of methanolic extract (Fig. 2C). However, no significant variation was observed in between the yield of EtOAc fractions of other sites (Figs. 2C, 3A,B). A standard curve was established using pure mahanine, ranges from 0 to 500 µg/mL using HPLC (Fig. 4A,B) to determine the mahanine content in EtOAc fractions of respective samples. Highest content of mahanine was found in samples collected from AP (228.5 ± 59.1 µg/mg) and the lowest was found in samples from AS (13.5 ± 5.4 µg/mg). The order of mahanine content in EtOAc fractions from different sites was AP > MH > DL > WB > AS (Fig. 4C).
Detailed weather analyses of the past 1 year and correlation with mahanine content. Weather conditions and rain fall records of medicinal plant cultivation sites could be one of the key factors for development of secondary metabolites 26 . To assess the relation of these conditions in the formation of mahanine in MK leaves, a detailed scrutiny of the last year's weather conditions at different sites were conducted. In AP and MH, where the mahanine content was maximum among the five selected locations, the average temperatures were 29.3 ± 2.6 °C and 25.2 ± 2.4 °C respectively for the 1 year preceding sample collection (Fig. S1A). The average humidity for those two sites were recorded as 67.7 ± 6.4 and 55.3 ± 21.1%, respectively (Fig. S1B) (Fig. S1A). Interestingly, the average temperature analyses showed, the more variance in temperature yielded less biosynthesis of mahanine in MK leaves. The record of average rain fall for these sites for the last 1 year showed that AS had ~ twofold higher rain fall than the highest mahanine yielding location AP (Fig. S1C) and the rainfall of low yielding site WB was not conspicuously different that of AP site. In the same time, the record of the approximate average age of the experimental plant had

Total micronutrient and heavy metal analysis in the soil samples and correlation with mahanine content.
In the present study, soil of AP-1 site showed the highest potassium (K) content and MH-4 site & MH-5 site showed lowest potassium (K) content among 25 soil samples (Table 2). In case of Na, soil of AP-2 site showed highest concentration and soil of WB-1 site showed lowest concentration. The Ca and Mg con-  (Table 1). Similarly, the concentration of Fe was highest in soil of MH-5 site and lowest in WB-1 site ( Table 1). Concentration of Co and Mn was found to be highest in soil of MH-4 site and lowest in AS-4, DL-4, and DL-5 sites (Table 1). Cd was  (Table 1).

Detailed physicochemical analysis of the soil samples and correlation with mahanine content.
The pH values were found to be highest in the soil of AP-2 site and lowest in the soil of AS-4 site ( Table 2). The average pH of the soil samples from AS was calculated as 6.1 i.e., acidic soil environment for MK plant growth. The soil samples from AP and MH had the average pH of 8.8 and 8.4, respectively and indicates alkaline environment for MK plant growth ( Table 2). The remaining soil samples of DL and WB had pH near neutral level. The electrical conductivity (EC) of different soil samples was found to be in the range of 54.8-694 µS/cm. The EC values were found to be highest for the soil of AP-4 site and lowest for the soil of AS-1 site ( Table 2). The total organic carbon and available phosphorus of soil samples ranged from 0.744 to 2.16% and 0.5-2.3%, respectively (Table 2). Available nitrogen in soil samples ranged from 0.294 to 0.785% (Table 2).

Microbial analysis of the soil samples and correlation with mahanine content. Based on obser-
vation of morphology of the bacterial colony, five different types of bacterial colonies were detected in the soil samples. The microbial plates that contains a minimum of 30-300 bacterial colonies were only considered and counted 23,24 . From the experimental data, no significant difference in the CFU was observed among all the soil samples tested from different locations. But, a trend of slightly high bacterial population (CFU) was observed in soil samples collected from AP and MH (6.7 ± 0.3 and 6.9 ± 0.3 log CFU) compared to other locations like AS and WB (6.5 ± 0.3 and 6.6 ± 0.3 log CFU) ( Table 3). The soils of AP and MH sites showed five diverse bacterial populations, whereas soil of AS sites contained only three types of bacterial colonies (Fig. S1F).

Discussion
The accumulation and/or formation of secondary plant metabolites helps the plants in germination, growth and survival from different perspectives 27 ; and those plant secondary metabolites have been the rich sources for the drug development research for various aliments since centuries 28 . Mahanine, a carbazole alkaloid isolated from the Murraya koenigii plant parts has been reported as important bioactive molecule for different health disorder like cancer and diabetes 2,7 . From our previous research findings, we have established for the first time that, mahanine is a potential drug candidate to treat breast cancer in subtype regardless manner 2 . During the isolation www.nature.com/scientificreports/ procedure of mahanine from MK leaves in our preceding study, we have observed a peculiar phenomenon that the yield of mahanine was significantly varied based on the geographical location of the plant sample collection. The present study has provided interesting perspectives on mahanine content in MK leaf that were collected from different geographical locations of India and along with the role of soil and weather conditions of those sites towards this phenomena. Mahanine content in MK leaves collected from AP sites were ~ 16.9 times higher and MH sites were ~ 12.4 times higher compared to that of MK leaves collected from AS sites. The average pH of the soil samples of AP and MH sites were found to be alkaline in nature (pH 8.8 and 8.4 respectively), whereas soil samples from AS sites were acidic in nature (pH 6.1). These finding suggests that soil alkalinity plays a significant role in the accumulation of higher level of mahanine in MK leaf. Accordingly, the electric conductivity (EC) of the soil samples also plays a significant role in formation of mahanine in MK leaves; plant mahanine content is directly proportional to electric conductivity. The mean EC of AP site (highest mahanine content) soils were 432 µS/cm, whereas for AS site (lowest mahanine content) soils were 78.3 µS/cm. Assam site has two times higher average rain fall than the AP sites. Higher rainfall leach down soil basic cations and make soil acidic and also it cause leaching of soluble salts down the soil profile reducing EC values. The high salinity of soil sample have crucial role in plant secondary metabolite formation 29 . MH sites, which are in a similar geographical region as the AP sites also had high soil pH and EC which in turn showed high mahanine content in MK leaf. The soil  www.nature.com/scientificreports/ microbiota also plays a crucial role in plant defense system and directly correlated with plant secondary metabolite formation 23 . Soil microbial analysis of this study revealed that more the value of log CFU of bacteria, greater was the concentration of mahanine in MK leaves. Interestingly, more diverse microbial population of bacteria was found in soil samples collected from AP sites, where the MK leaf mahanine content was highest compared to that of AS sites, where lowest soil microbial population as well as mahanine content. Although, single plate count microbial assay does not provide entire range of soil bacterial diversity, there was an indication of relation between bacterial diversity and mahanine content. A detailed culture independent mechanistic bacterial diversity study can depicts the role of microbe(s) in secondary metabolite (in specific mahanine) formation in MK leaves need to be investigated. Other micronutrient of soil such as Na, K, Ca and Mg are also reported to play important role in formation of specific type of plant secondary metabolites 21 . These nutrients in soils of AP and MH sites were found significantly higher amount compared to those of WB or AS sites. The other factors such as temperature, humidity and plant's moisture content were found to have no significant relation with the mahanine content of MK leaf. Altogether, the present study provides the evidence of variation in mahanine in MK leaves depending upon the soil physicochemical and biological parameters, the direct and interactive contribution of each factors cannot be accounted from this data. This study warrant for research under controlled environment to establish quantitative relationship between different weather and soil parameters and mahanine content in MK leaf to develop agro-technology in enriching a specific bioactive metabolite.

Data availability
All data generated or analysed during this study are included in this published article (and its Supplementary Information files).