Rheum species, commonly called rhubarb, are included in endangered plants list and are under great threat. It has been listed as vulnerable by various agencies like IUCN, UNEP and WWF particularly from Kashmir Himalaya [1]. Rheum has sixty species all over the world and mostly famous for its medicinal value, as recent studies have proved rhubarb as one of the anticancer plant. Rheum includes perennial, stout herbs, mainly distributed in the temperate and sub- tropical regions of world chiefly in Asian countries viz. India, Nepal, Bhutan, China, Pakistan, Korea, Turkey, Russia and Tibet. Many compounds used in today’s medicine have a complex structure, and synthesizing these bioactive compounds chemically at a low price is not easy [2]. The age old traditional values attached with the various forest types and the varieties of forest products (i.e., medicinal plants) have gained tremendous importance in the present century [3]. In china, Rheum plant is worshipped as it cures so many diseases and it is called as Dahuang in China. The Rheum plant is an eatable plant. It can be taken as a food ad it can be also cooked. Its juicy stalks are eaten raw and its leaves are cooked as a vegetable. In India it is found in Western Himalaya, and Northern Himalaya. In Kashmir it is found in all in over the Kashmir Himalaya which includes regions of Ladakh, Guerz, Anantnag and Baramulla districts. R. webbianum is used in treatment of indigestion, abdominal disorders, boils, wounds and flactuaence [4]. It is also helpful in managing cancers [5]. R. emodi is puragative, stomachache, astringent, tonic and effective in curing skin diseases [6,7] it is an antioxidant [8] and [9] cytotoxic in nature [10], Kinase II inhibitory [11], anti-viral [12] and nephroprotective [13]. Moreover, it is used as textile dyeing [14], antimicrobial, anti-tumor, anti-inflammatory [15] used in cosmetics ad as food colorant [16], live stimulant, purgative, anticholesterololeamic, antitumor, antiseptic, tonic, and, antiparlinson [17]. Hypatoprotective principles that can prevent and treat liver damage [18], blocks the binding of SARS-CoV S proein to ACE2 and infectivity of S protein pseudotyped retrovirus to vero E6 cells [19], Antidiabetic, similar to insulin [20], Nephroroprotective properties [21] anticancer, antioxidant [22,23]. R. spiciforme is also an adulterant and also used also in the treatment of boils, wounds, rheumatic pain. Roots are frequently used for the treatment of bone fractures, backache and joints pain [24]. Chromatography is one of the fast emerging tools by which the quality control and fingerprint of herbs can be assessed accurately. Using this technique, the identification of various chemical markers of the herbal drugs can be easily done and it also helps to identify the specific herb in combination of other herbs. Popularity of HPLC for analysis of herbal drugs is due to its economic, rapid and simultaneous screening of large number of herbal samples in less time. The main active ingredients of the Rheum species are a series of anthraquinones, dianthrones, glycosides and tannins. The anthraquinone derivatives including emodin, aloe-emodin, rhein, physcion, chrysophanol and their glucosides are the accepted important active principles. Rhaponticin, a distyrene derivative, only exists in non-quality (inferiorgrade) rhubarb. In quality rhubarb and most exported rhubarbs, the content of rhaponticin is not detected. Like all such substances, rhein is a cathartic. Rhein is commonly found as a glycoside such as rhein-8-glucoside or glucorhein. Rhein was first isolated in Yu et al. [25]. Originally the rhubarb plant which contains rhein was used as a laxative. It was believed that rhein along with other anthraquinone glycosides imparted this activity. Rhein has been reevaluated as an antibacterial agent against Staphylococcus aureus [26]. Present study was aimed to find out the anthraquinones from the two Rheum spp. for which three standards were used i.e., aloe emodin, emodin and rhein.

The roots and rhizomes of R. webbianum and R. spiciforme were kept in brown paper bags dried under room temperature. The in vitro explants i.e., leaves, roots and callus [27] of these species were also dried in room temperature in paper bags. The dried roots and rhizomes of all these species were grinded in pestle and mortar to powder form for making methanol (HPLC grade) extracts. The glassware and methanol (HPLC grade) was procured from commercial suppliers. Triple distilled water was used in the laboratory for different steps.

Extraction of plant material

The powdered roots and rhizomes of each sample (30 g) were charged in a soxhlet apparatus and extracted with 500 ml of HPLC methanol on water bath. The extraction was continued for one week. The extract was concentrated and dried on rotary evaporator under reduced pressure. The resultant semisolid, sticky extract of each sample was stored at 4ºC till further analysis. Each extract was subjected to phytochemical screening to detect the different types of constituents present in it. We used Dragendorff’s test for alkaloids, Fehling solutions test for carbohydrates, Millions test for proteins and amino acids, Salkowski reaction for steroids, Shinoda test for Flavonoids, Keller-Killiani test for glycosides, FeCl3 test for tannins and phenolic compounds and Sudan Reagent test for Fats and oils.

HPLC analysis

The chromatographic analysis was carried out on a Waters HPLC system comprising binary pumps (515), auto injector (2707) and PDA detector (2998), controlled by Empower Pro software. Each standard marker (rhein, emodin, aloe-emodin) and extract (1 g extract dissolved in 5 ml methanol) was chromatographed on a C₁₈ column (250 mm × 4.6 mm; Sunfire) with gradient elution by using methanol (mobile phase A) and 2% acetic acid (mobile phase B) at a flow rate of 0.5 ml/min. The column was maintained at a temperature of 30ºC. The injection volume was fixed at 10 μl and LC-UV chromatographs were extracted at 254 nm. The gradient program for elution is given in Table 1. Purity of each marker peak in LC-UV chromatogram of each sample was ascertained by PDA analysis.

Table 1: Estimation of different compounds from methanolic extracts of R. webbianum.

For quantification of the three markers (aloe-emodin, rhein & emodin), a standard solution containing these three markers (1 μg/ml) in methanol was prepared and analysed (n=6) using the optimized HPLC methanol. Peak area of each marker (mean ± SD) was determined, and it was used to calculate the content of markers in samples of Rheum collected from different sources. For sample analyses, each extract obtained after recovery of the solvent was dissolved in 150 ml of methanol. This extract solution was analysed by the HPLC method and contents of markers were calculated as follows

Content of markers in (μg/30 g) = (A_S/A_T) × l × 150:

Where, A_S = peak area of marker in standard solution A_T = peak area of marker in extract solution 1= concentration of marker in standard solution in 150 μg/ml = dilution factor.

Rheum webbianum

The phytochemical screening of extracts of R. webbianum showed that the amino acids were absent in the samples of Panzila Top, Khardungla and Tangsti, whereas glycosides were absent in the samples of Khardungla and Tangsti. It was also observed that all types of phytoconstituents were present in each of the in vitro explants also (Table 1).

HPLC analysis

The contents of aloe-emodin, emodin and rhein in various samples of R. webbianum were found to be in the range of 67.5-4500, 255-2385 and 0.4-1143 μg/30 g of the plant material, respectively (Table 2). The LC-UV chromatograms of extracts of different samples of R. webbianum along with in vitro explants are shown in Figure 1. The roots showed the presence of anthraquinones i.e., aloe-emodin, emodin and rhein in all the observed samples. The maximum content of aloe-emodin and rhein was found in Panzila Top sample, whereas maximum content of emodin was found in Tangsti sample (Table 2 and Figure 2). Of these, Panzila Top, populations (2n=22), whereas all other populations are tetraploid (2n=44). The amount of aloe emodin and rhein is found to be very high in diploid cytotype, compared with all the populations of tetraploid cytotypes. However, the amount of emodin is more than two populations and less than other two populations. The leaves, roots and callus of in vitro explants were also analysed for anthraquinones content (Table 2 and Figure 2). The maximum content of the markers of aloeemodin and rhein was found in in vitro callus, while the maximum content of emodin was found in in vitro leaves (Table 2, Figure 2).

Figure 1: (A-H): LC-UV chromatographs of R. webbianum samples collected from Tangsti (A), Khardungla (B), Pazila top (C), Parakhchik (D), Tangole (E), In vitro leaves (F), In vitro roots (G), In vitro callus (H).

Figure 2: Content of anthraquinone markers in R. webbianum samples of wild and in vitro raised plants.

Table 2: Content of different active constituents in various samples of wild R. webbianum populations and in regenerants of in vitro raised plants.

Rheum spiciforme

Phytochemical screening: The extracts along with the in vitro explants of R. spiciforme were screened for phytochemical tests, and it was observed that all the compounds i.e., amino acids, steroids, glycosides, etc., were present in all the samples (Table 3).

Table 3: Estimation of different compounds from methanolic extracts of R. spiciforme.

HPLC analysis: The sharp and symmetrical peaks were observed for all the three marker anthraquinones i.e., aloe-emodin, emodin and rhein in all the samples of R. spiciforme (Figure 3 and Table 3). The maximum content of aloe-emodin (3409.5 μg/30 g) and rhein (531.4 μg/30 g) was found in Chakwali, whereas, maximum content of emodin (915.0 μg/30 g) was found in Dahi Nala. The different parts of in vitro explant i.e., leaves, roots and callus were also analysed for markers. The maximum content of aloe-emodin and emodin was found in in vitro callus whereas rhein was absent in all the three explants (Table 4 and Figure 4).

Figure 3: (A-I): LC-UV chromatographs of R. spiciforme samples collected from (A) Chakwali, (B) Dahinala, (C) Dawar hills, (D) Habba Khatoon mountain, (E) Satni mountain, (F) Tragbal, (G) In vitro leaves, (H) In vitro roots, (I) In vitro callus.

Figure 4: Content of anthraquinone markers in R. spiciforme samples of wild and in vitro raised plants.

Table 4: Content of different active constituents in various samples of wild R. spiciforme populations and in regenerants of in vitro raised plants.

Plants are rich source of effective and safe medicines due to presence of different bioactive molecules such as alkaloids, flavonoids, glycosides, tannins, phenolic compounds, etc. [28,29]. Rheum is a well-known medicinal plant having anti-cancer and anti-oxidant activities. Anthraquinone is the major class of phytochemicals, which is responsible for its pharmacological activities. These constituents are mainly present in roots and rhizomes. The main members of anthraquinone class include aloe-emodin, emodin, chrysaphanol, physcion and rhein, which are proved as anticancer agents [30]. Anthocyanins and flavonols are also found in Rheum [31,32]. In the present study, three anthraquinones were analysed i.e., aloe-emodin, rhein and emodin. Aloe-emodin (1,8-Dihydroxy-3-(hydroxymethyl)-9,10-anthraquinone) has been reported to exhibit anticancer activity on neuroectodermal tumors, lung squamous cell carcinoma and hepatoma cells [33]. Emodin (1,3,8-trihydroxy-6-methylanthraquinone) is an active constituent of many herbal laxatives. It has been used for the treatment of inflammatory diseases such as peptic ulcers and as a laxative, and others such as skin burns, gallstone, hepatitis, inflammation and osteomyelitis, etc. [34]. Rhein (1,8-dihydroxy-3- carboxyl anthraquinone) is also known as cassic acid, is a substance in the anthraquinone group obtained from rhubarb. Rhein is a cathartic and is commonly found as a glycoside such as rhein-8-glucoside or glucorhein. Rhein was first isolated [25]. Originally the rhubarb plant which contains rhein was used as a laxative. It was believed that rhein along with other anthraquinone glycosides imparted this activity. Rhein has been reevaluated as an antibacterial agent against Staphylococcus aureus in 2008 [26].

In the present study, the two species of Rheum i.e., R. webbianum and R. spiciforme were screened for the presence of different phytoconstituents. These were collected from the different parts of Kashmir (India) at different altitudes and were also grown in vitro. There were total 17 samples, including the in vitro explants of each species from which the anthraquinones were derived. In case of R. webbianum, amino acids were absent in the sample collected from Panzila Top, Khardungla and Tangsti. Glycosides were absent in Khardungla and Tangsti samples. All these compounds were also present in in vitro explants. Raashid [35] has also reported the similar phytochemical behavior for R. webbianum. All these constituents i.e., alkaloids, proteins, flavonoids, carbohydrates, amino acids, steroids, glycosides, tannins and phenols, fats and oils were also present in all the samples of R. spiciforme, as well as in the in vitro raised explants. These results indicate that there is no uniform effect of altitude on the presence of phytochemicals in any of the two species. In present studies, the glycosides were screened in roots of Rheum species and also from in vitro explants (roots, callus and leaves). All samples of R. webbianum were also analyzed for the content of these three markers. All the three markers were present in all samples of R. webbianum. The maximum content of aloe-emodin (4500 μm/30 g) and rhein (1143 μm/30 g ) was found in sample collected from Panzila Top whereas maximum content of emodin (2385 μm/30 g ) was found in Tangsti sample. Rhein was also present in all these samples. In the case of in vitro explants, the maximum content of aloe-emodin (48 μm/30 g) was found in in vitro callus, the maximum content of emodin (13 μm/30 g ) was found in in vitro leaves, rhein was absent in in vitro roots and leaves, whereas rhein (10 μm/30 g) was only present in in vitro callus. The amount of these anthraquinones and their derivatives has also been studied in many other species of Rheum such as R. tanguticum [36]. R. officinale [37], R. palmatum [38] and R. ribes [39] etc. (Supplementary Table S1).

Supplementary Table 1: Gradient elution programme.

The nine samples of R. spiciforme were also analyzed through HPLC for the quantification of the markers. All of these markers were present in each sample of R. spiciforme. The maximum content of aloe-emodin (3409 μm/30 g ) was found in Chakwali, whereas, maximum content of emodin (915 μm/30 g ) was found in Dahi Nala sample. The maximum content of rhein (531 μm/30 g) was also found in Chakwali. It was also observed that rhein was present in all the samples of R. spiciforme, whereas it was absent in some of the samples of R. emodi. In case of in vitro explants, it was noted that the maximum content of aloe-emodin (31.35 μm/30 g ) and emodin (22 μm/30 g ) was found in in vitro callus whereas rhein was absent in all these three explants.

It was observed that maximum aloe-emodin, emodin and rhein were found in R. webbianum as compared to R. spiciforme. The maximum content of aloe-emodin and rhein of the in vitro explants was also found in R. webbianum, whereas rhein was absent in explants (in vitro) of R. spiciforme. Only maximum content of emodin was found in in vitro explants of R. spiciforme.

Present study is the first report on R. spiciforme from India for phytochemical screening as no previous work was done in India and it was also observed that the R. webbianum contains highest content of these three anthraquinones and it was also observed that in R. webbianum has the amount of active compounds present in tetraploid plants was less than diploid plant. Similarly, the amount of active principle is reported to be more in the diploid than polyploids by some other workers [40-42]. Generally the amount of these active principles are found to be more in polyploids as compared to diploids [43,44].

It was observed in the present study, that the amount of these compounds show great variation, to check the variation we must go for authenticated plants and must explore extensively area to know the best chemotype plant. The value of contents must be varying due to genetic diversity and/or ecological factors, so we must mark out the best genotype growing in specific type of environment which is having the maximum amount of these active principles, only then we can have the standardized drug with specific amount of the active principles. Also, the amount of these active principles is known to vary with the age of plants, so we must study the amount of these active principles in the cultivated plants at different stages of age. The tissue cultured plants show less amount of active principles as compared to wild grown plants which might be due to the young age of tissue cultured plants as they were hardly 4-5 months old whereas the wild plants were very old, as the amount of active principles increase with increase in age of plants, so the age differences between tissue cultured and wild grown plants might showed the variation in content of active principles. Correlation in amount of secondary metabolite with altitude has been reported in R. emodi [7,45-46]. Prasad and Purohit [7] reported the concentration of active constituents and calorific value of R. emodi and R. nobile in Sikkim Himalaya. In their study, high calorific values was recorded in R. nobile in comparison to R. emodi, and active constituents of both the plant species were found to decrease in low-altitudes conserved plants compared to the wild plants. But in the present study, it was found that no co-relation between altitude and content of markers was found. This lack of correlation may be attributed to the other factors such as age of the plant and magnitude of exposure of the plant to sunlight also affects concentration of markers in the plants.

Phytochemical screening showed the presence of amino acids, alkaloids, flavonoids, steroids, fats and oils, tannins and phenols, carbohydrates and glycosides in different samples of Rheum species. In some samples of R. emodi, amino acids, steroids and glycosides were absent. In R. webbianum, constituents like alkaloids, proteins, steroids, etc., were also present. Only amino acids and glycosides were absent in two of the samples, whereas all the compounds were present in in vitro explants of R. webbianum. In R. spiciforme, all constituents i.e., alkaloids, proteins, flavonoids, carbohydrates, amino acids, steroids, glycosides, tannins and phenols, fats and oils were also present in all the samples as well as in samples of in vitro explants. These species of Rheum were also screened to quantify the anthraquinone markers (aloeemodin, emodin and rhein). The methanolic extracts were analysed to determine the content of aloe-emodin, emodin and rhein by HPLC method. All the three anthraquinones i.e., aloe-emodin, emodin and rhein were present in R. webbianum, but in case of in vitro explants of R. webbianum, rhein was present only in callus. The highest amount of aloe emodin (4500/30 g), emodin (1500.0/30 g) and rhein (1143.0/30 g) was observed in Panzila Top, Khardungla and Panzila Top samples respectively. In R. spiciforme highest content of aloe-emodin (3409.5/30 g) and rhein (531.4/30 g) was found in Chakwali samples, while highest content of emodin (915.0/30 g) was present in the samples of Dahi Nala. Rhein was absent in all in vitro explants of this species. Among all the three species, the maximum contents of aloe-emodin, emodin and rhein were found in samples of R. webbianum and amongst the in vitro explants, the maximum content of aloe-emodin and rhein was found in R. webbianum and that of emodin was observed in R. spiciforme. The observation of present study showed that R. webbianum is most abundant and rich in contents of active compounds. The results of present study can be efficiently utilized in future research programmes for the selection of elite material and for devising the utilization strategies for best chemotypes.

The authors hereby declare that they have no conflict of interest.

All authors equally participated in designing experiments analysis and interpretation of data. All authors read and approved the final manuscript.

This study was supported by DST, GoI, New Delhi funded women entrepreneurship project, the assistance of which is highly acknowledged.