Agro-climatic zone-based identification of elite Terminalia arjuna accessions concerning to arjunolic acid production

Man has been dependent on vegetation in different forms since antiquity. Plants and trees have been used in the form of medications since the old times. Although the use of plant parts in the form of medicine has been less than that of the plants, therefore today it is necessary to explore some such medicinal trees. Among these, one of these trees is Arjun, whose bark is described also in Ayurveda due to its divine medicinal properties in heart diseases. The present study based on the isolation and analysis of the Phyto-constituents of the stem bark extract of Terminalia arjuna member to the family Combretaceae collected from different agro-climatic zones of India. The samples were subjected to quantitative phytochemical analysis i.e., arjunolic acid, screening by implementing the standard procedure. Observation has shown the presence of arjunolic acid in the stem bark extracts in different fractions obtained by the use of various organic solvents. Therefore, the bark extracts of the chosen plants may function as a good source of components of useful drugs for cardiovascular disorders and may also be used for the preparation of other pharmaceutical products.


Introduction
Men have been using plant-based medicines since time immemorial. Almost every civilization has a history of using medicinal plants. According to Ncube et al. [1], Medicinal plants are the prerequisite for the traditional systems of medicines, pharmaceutical industries for synthetic drugs. In ancient times, Egyptians, Indians, Chinese, Africans and others used a variety of plant products for curing all kinds of ailments [2]. The hypocholsteremic, hypolipidemic, anticoagulant, antihypertensive, antithrombotic, antiviral, antifungal and antibacterial activities of arjunolic acid were seen by the Tripathi and Singh [3] and Pettit et al. [4] in their findings. The effectiveness of arjunolic acid in many cardiac disorders like angina, myocardial infraction, hypertension, hypercholesteremia, cardiac arrest etc. were reported by the Rose and Treadway [5] and Khan and Balick [6]. Other studies also reported that it's bark retains significant hypotensive effect, increasing coronary artery flow and protecting myocardium against ischemic damage and mild diuretic, antithrombotic, prostaglandin E (2) enhancing and hypolipidemic activity of arjunolic acid the experimental findings by the Dwivedi [7].
Thus, medicinal plants are the local heritage with global importance. The number of flowering plants on the earth estimated presently is about 2, 50,000 species of which nearly 70,000 species are used for medicinal purposes, both in developed and developing countries [8]. An estimate suggests that about 13,000 plant species are known to have worldwide use as drugs.
It is reported that 41% prescription in the USA and 50% in Europe contains constituents from natural products.
Phytochemical tests have been performed on about 5000 species; and nearly 1100 species are extensively exploited in 80% of Ayurvedic, 46% of Unani and 33% of Allopathic medicines [9]. India has 2.4% of the world's land area with 8% of global biodiversity and harbors one of the 12-mega diversity centers, having over 45,000 plant species. Its diversity is unmatched due to the presence of 16 different agro-climatic zones, 10 vegetative zones and 15 biotic provinces [10]. The country accounts for 17,500 species of higher plants. Of these, more than 2000 documented species exhibit medicinal value vis-à-vis 1100 species being used in different systems of medicines.
International trade in medicinal plants and phytopharmaceutical preparations is a major force in the world economy. Their demands are increasing both in developing and industrialized nations. A report prepared by the Export-Import Bank of India has estimated that the international market of medicinal plant-related products is in the range of US$ 107 billion with an annual growth rate of 7% (According to Global Summit on Herbals & Natural Remedies, Chicago, USA). According to research from global Industry Analysis, the global herbal supplement and remedy industry has been estimated to be $ 107 billion by 2017. India's share in the global herbal market in the year 2017 is of the order of 2 billion dollars. India is the second-largest exporter, next to China. According to the current estimation, phytomedicines used in health care globally by 40% of the total population [11].
A different system of treatments such as ayurvedic, Unani, homeopathy and siddha originated in India. India is the birth-place of the renewed system of indigenous medicine such as Unani, Ayurveda, Homeopathy and Siddha. In India, nearly 95% of the prescriptions were planted primarily based on ancient systems [12]. In this study, the quantitative estimation of Phyto-constituents of stem bark of Terminalia arjuna commonly referred to as "Arjun" (fam-Combretaceae), was done for the presence of arjunolic acid.
The study was conducted to assess the natural variation of Terminalia arjuna to investigate the populations along with the natural habitats of this medicinal plant for the phytochemical contents upon comparison from nine states and five different agro-climatic zones of India. The main scope of this experiment was the selection of the Terminalia arjuna accessions with a high content of arjunolic acid from the various population collected from different agro-climatic zones.

Collection and processing of plant samples
Plant samples (mapping population) of Terminalia arjuna were obtained from All India germplasm of arjun established between 2001 and 2004 at Central Tasar Research and Training Institute (CTRTI), Nagri, Ranchi that maintains superior arjun accessions from nine states, viz., Andhra Pradesh, Assam, Chhattisgarh, Jharkhand, Maharashtra, Madhya Pradesh, Orissa, Uttrakhand and Uttar Pradesh altogether representing five agro-climatic zones, viz., Eastern plateau and hill (EPH) regions, Southern plateau and hills (SPH) region, Eastern Himalayan (EH) Region, Western Himalayan (WH) region and Central plateau and hills (CPH) region (Fig. 1). For the selection of various accessions of Terminalia arjuna tress, some criteria were chosen: the tress growth should be vigorous, healthy and showing superiority in height and diameter i.e., straight, cylindrical, non-forking, non-twisting bole when compared with surrounding trees and resistant to pests and diseases. The distance between the collections of two accessions must be at least 200 m from their natural habitat. A total of 140 accessions were sampled for bark patch (10 cm x 10 cm) at 1.34 m diameter breast height (DBH) for the estimation of arjunolic acid content. The details of the accessions of arjun represent different agro-climatic regions bark are given in Table 1.

Chemicals
Only HPLC and/or analytical grade chemicals and reagents (Sigma, USA; Himedia, USA; Merck, Germany, etc.) were used during the present study.

Collection and processing of bark patch
A bark patch of 10 cm x 10 cm was sustainably removed at 1.34 m from each of 140 accessions of Terminalia arjuna. The thickness of the sampled bark patch was measured at several places by Vernier calliper. The bark was washed with distilled water, followed by the estimation of its fresh weight. Subsequently, the bark patch was shrouded and cut into the pieces and dried at room temperature in the shade and watched regularly to reduce the attack of fungi. It took a week or so until attaining a constant dry weight that measured and recorded. A fine powder of dried bark patches was made with the help of grinder and sieved through a 25 mm fine mesh and divided into three parts representing as replicates for extraction and estimation of arjunolic acid and stored in air-tight containers with necessary markings for identification and kept in a cool, dark and dry place for further use.

Extraction of arjunolic acid from bark powder
200 mg bark powder was exactly weighed and placed in a conical flask (100 ml) and then mixed with 20 ml of ethyl acetate. All conical flasks were retained for 10 min for presoaking as pre-leaching. It was followed by an extraction process that was performed by warming the content at 65 °C for five min, i.e. below boiling point (71.1 ºC) of the ethyl acetate in a 600 W microwave oven. An irradiation treatment was given to the samples under microwave for one minute followed by cooling for 1 min between two irradiations. Whatman filter paper no.1 was used for the filtration of suspensions. The residue was washed twice with ethyl acetate. The washings were pooled with the filtrate and the solvent was vacuum evaporated at 40 ºC (Vacuum Oven, TEMPO Instruments Pvt. Ltd) and lyophilized. The solvent-free residue was dissolved in 1 ml HPLC grade methanol and transferred to 1.5 ml Eppendorf tubes and stored at −20 °C in the deep freezer until estimation of arjunolic acid on HPTLC.

Arjunolic acid standard
A pure sample of arjunolic acid (MW 488.70, Melting point 296 °C, purity > 99%) as one mg powder (vial) was purchased from Sigma-Aldrich India, was used in the concentration 1000 µg/ ml. The working standard solution of 100 µg/ml was prepared from the standard stock solution by drawing a known volume of the latter and diluting in the ratio of 1:10 by HPLC grade methanol. A volume of the working standard (arjunolic acid) solution equal to that of the bark sample was loaded alongside the latter on the HPTLC plate. The peak area of the standard arjunolic acid was used for the computation of arjunolic acid in the bark sample extract.

HPTLC processing and estimation of arjunolic acid in the bark extract
HPTLC processing needs a clean pre-coated and activated silica aluminum plate, a micro-syringe (dispenser) for loading standard solution/ sample extract, oven, twin trough

TLC plate specification and processing
HTPLC silica gel plates of dimension 20 cm x 10 cm (60 F 254, Merck, India) with specifications given in Table 2 were used. The plates were marked with a pencil for the direction of development and developed with 20 ml methanol per trough in a 20 × 10 cm TTC to the upper edge. Subsequently, they were dried in a clean drying oven at 100° C for 20 min and allowed to equilibrate with lab atmosphere (temperature, relative humidity) in a suitable container free from dust and fumes. The heat treatment was given to the plates at 120 °C for half an hour for activation before the use and held either on both side edges or on the top edge. Most of the time, the plates were used without pretreatment unless chromatography produced impurity fronts due to their contamination.

Loading standard compound and sample extract on the plate
An 8 µl standard working stock (100 ppm arjunolic acid) or 5 µl bark extract (equivalent to 1 mg dry bark powder) was loaded on the plates with help of CAMAG Linomat 5 applicator whose parameters are given in Table 3. The samples  were applied as bands by spray-on technique following the scheme given in Table 4.

Chromatogram development for arjunolic acid
Plates were developed in a saturated 20 cm x 10 cm TTC. The chamber was opened and a piece of the filter of size 20 cm x 10 cm was placed in the rear through. A 20 ml of freshly prepared mobile phase/developing solvent (Composition: 60 ml toluene, 30 ml ethyl acetate, 1 ml formic acid and methanol 10 ml) was poured into the chamber prepared so that the filter paper thoroughly wetted and adhered to rear TTC wall. Subsequently, the chamber was tilted to the side (about 45°) so that the solvent volume in both troughs got equalized. The chamber was placed on the bench and replaced the lid, allowing for 20 min for its equilibration.
The preferred developing distance (60 mm from the lower edge of the plate) with a pencil on the right edge of the plate was marked with the lid having been slide off to the side for the insertion of the plate into the front trough. The plate was adjusted in such a way that its layer faced the filter paper and it's back rested against the front wall of the TTC. The lid was replaced and the plate was developed to the mark. It was followed by the removal of the plate from TTC by opening the lid. The plate was dried vertically in direction of chromatography in a stream of cold air for 5 min. After each development, the residual mobile phase and filter paper were discarded. Before being prepared for the next run, the chamber was dried and, if necessary, also cleaned.

Derivatization of arjunolic acid in the chromatogram
The loaded sample on the HPTLC plate was derivatized by immersing for 20 min in the dip tank device containing 200 ml derivatization solvent reagent (Composition: 85 ml ice-cold methanol, 10 ml acetic acid, 5 ml sulphuric acid and 1 ml anisaldehyde). The plate was subsequently removed from the tank device and allowed the excess reagent to drip off. The back of the plate was wiped off with tissue paper. It was followed by drying of the plate at 100 °C for 2-5 min in the hot air oven.

Visualization of the developed chromatogram and computation arjunolic acid content in the bark extract
The derivatized sample fingerprints (chromatograms), along with standard arjunolic acid chromatogram of a known quantity, obtained by derivatization was scanned at 595 nm on CAMAG scanner (visual; Fig. 2) with the specification given in Table 5. A software program supported the scanner for start/endpoint for scanning plates, baseline correction, resolution and coverage peak area, whose specification is mentioned in Table 6. The optical density of the sample bands was compared with that of the standard band developed from loading known quantity of pure arjunolic acid for computation of arjunolic acid content in the corresponding bark samples (Figs. 3, 4, 5, 6). Let the peak area for bark sample be "x" and for pure arjunolic acid be "y". The arjunolic acid content in bark sample (B) be computed from the following expression:

Arjunolic acid content
Arjunolic acid in the bark samples collected from one hundred and forty Terminalia arjuna accessions, belonging to nine states and five agro-climatic zones, assembled as germplasm bank was estimated. The arjunolic acid content was expressed as a percentage. The highest percentage of arjunolic acid in the extracts was found to be 0.54% in accession UKDDLP and the lowest percent extractive value 0.003% was estimated in the accession APMD03, methanol fraction of Terminalia arjuna stem (1) B(μg∕mg bark dry weight) = 0.8x/y bark was used. The percentage of arjunolic acid in the extracts was ranging from 0.003% to 0.54%. The percentage of arjunolic acid the stem bark of arjun is given in

Arjunolic acid (%)
Arjunolic acid (%) was significantly (p < 0.05) affected by state and T. arjuna accessions. The agro-climatic zone did not influence arjunolic acid (%). Accessions from the Uttrakhand (UK) state exhibited significantly the highest value for arjunolic acid. On the other hand, the accessions from Assam (AS) state had significantly the lowest value for arjunolic acid (%). The accessions from UK state registered 238% higher value for arjunolic acid content than accessions from AS state (Fig. 7I).
The sampled accessions exhibited significant (p < 0.05) variation in the arjunolic acid (%) in their barks. Accession UKDDLP belonging to Uttrakhand state and WHR agroclimatic zone registered the highest value and accession APMD03 belonging to Andhra Pradesh and SPH agro-climatic zone, the lowest value for the arjunolic acid (%). The arjunolic acid increment was 17,900% in accession UKD-DLP over accession APMD03 (Table 7).

Discussion
A species survives through various populations, which become discrete in space and time due to the combined influence of intrinsic genetical changes and extrinsic    and generate variability and differentiation. The extrinsic geo-climatic selection forces are in fact drivers for adaptation and conservation of specific genetic pattern (s) of the population. Therefore, the dataset for populations needs to be analyzed taking these considerations.
In most of the traditional systems of treatment, the different parts of the plants used in the treatments for different ailments by the local peoples of those particular area. Plants synthesize metabolites during their biosynthetic pathways, these metabolites used by the plants called primary metabolites. Further, plants produce secondary metabolites mostly in stress conditions and used them for defensive purposes. These secondary metabolites accumulate in specialized vesicles or organs in the plant body. Phenolic compounds, alkaloids, terpenoids, volatile oils are some example of secondary metabolites. These secondary metabolites used in different purposes in the treatments of different diseases and disorders [13]. Consequently, the globe Health Organization (WHO) established a definition of medicinal plants: "A medicinal plant is any plant or plant part that can be used for the therapeutic purpose or that may be a precursor for the synthesis of helpful drugs" [14]. Moreover, the United Nations agency (World Health Organization, Geneva.2000) defines the medicinal plant as seasoning preparations made by subjecting plant materials to extraction, fractionation, purification, concentration or different physical or biological processes that can be made for immediate consumption or as a basis for seasoning merchandise. Medicinal plants are those plants that contain active ingredients used to cure the disease [15].
A large number of secondary metabolites produced by the plants that do not directly involve in primary processes such as growth and development. Secondary metabolites comprise three chemically distinct groups, i.e. terpene phenolic compounds and N-containing compounds (strychnine, nicotine, caffeine, cocaine, capsaicin). For many years, they have been presumed to be by-products or metabolic wastes and do not participate in physiological functions. They have now been recognized to perform plant defense functions and involve in signal transduction, playing a major role as the deterrent to adverse conditions and microbial/ pest attacks. For example, high tannin content in sorghum cultivars conferring astringency and poor digestibility discourage attack by birds [16]. High salicylic acid content confers adaptability and pathogen resistivity to plants [17][18][19][20].
Arjunolic acid along with other secondary metabolites like phenols and tannins accumulates in the bark and possibly confer resistance against natural vagaries and insect pest attacks. Interestingly, the compound has been extensively investigated for pharmaceutical purposes rather than its biological functions in the host plant, i.e. T. arjuna. Arjunolic acid has also been reported from other plants such as Cochlospermum tinctorium, Cornus capitata, Leandra chaetodon, Combretum leprosum, Campsis grandiflora, Syzygium guineense, Combretum nelsonii [21]. Like Vertical lines represent the standard deviation (SD). Data are mean of three replicates and significant at p < 0.05 morphometric traits, arjunolic acid content in the bark also exhibits a great variation across accessions, locations and agro-climatic zones. Chemically, arjunolic acid is triterpenoid saponins of great therapeutic value but its biosynthesis pathways are obscure. Consequently, the observed variability of its content across accessions, locations and agro-climatic regions may be attributed to the interaction between genotype and geo-climatic condition. In consonant, accession UKDDLP from Uttrakhand and Western Himalayan agro-climatic region produces the highest amount of arjunolic acid and accession APMD03 from Andhra Pradesh of Southern plateau & Hill agro-climatic region, the lowest amount of arjunolic acid. The plausible reason appears to be the temperate region and high annual rainfall of 1500 mm in Uttrakhand in comparison to arid condition with 50-100 mm average annual rainfall in Andhra Pradesh. The present study endorses that geoclimatic variables like environment, habitats, geographical conditions, altitude etc. have reflective and reproducible effects on the quantitative content of arjunolic acid in T. arjuna.
In the literature, the investigations have been devoted to the extraction and purification of arjunolic acid from the bark of T. arjuna employing HPLC or HPTLC systems [22]. In the present investigation, the HPTLC procedure has been adopted for quantification of arjunolic acid in the bark of 140 accessions of T. arjuna that has indeed been done for the first time on such a large scale. Our results bring out a great opportunity for field selection of superior accessions of T. arjuna for obtaining the high yield of arjunolic acid. The accessions belongs to the Uttrakhand state UKDDLP in India and were found to be with the highest content of arjunolic acid in their stem bark when compared to accessions of Andhra Pradesh. These accession may be introduced for large-scale plantation for commercial extraction of arjunolic acid on a sustainable basis.

Conclusions
Terminalia arjuna is a plant with various pharmacological activities. Different parts of the plant is used for different activities. The most widely used part of the plant is the stem bark. The present study was aimed to investigate the phytochemical screening with the help of HPTLC that revealed the presence of high arjunolic acid content in different accessions of Terminallia arjuna bark extract from Uttrakhand in India. Thus in conclusion: • The accessions from the Uttrakhand state and Western Himalayan region agro-climatic zone were found with the high content of arjunolic acid. • This triterpenoid saponin i.e., arjunolic acid has been reported for the treatment of cardiovascular disorders as well as some other diseases. • However, further studies are required to separate the compound of interest in purified form from the partially purified plant extracts for the preparation of medicines. Pharma industries have a big demand for arjun bark as it used in different pharmaceutical formulations. • The identified accessions with high arjunolic acid content in the bark need to be incorporated in the genetically improved clones and introduced to the commercial plantation for high return and conservation of natural resources of T. arjuna."