Micropropagation studies and phytochemical analysis of the endangered tree Commiphora wightii

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Micropropagation studies and phytochemical analysis of the endangered tree Commiphora wightii Ch. Mohan a , B. Naresh a , B. Kiran Kumar a , Veena Reddy b , P. Manjula a , B. Keerthi a , D. Sreekanth a , Syeda Fatima Manzelat c , Prathibha Devi Cherku a,∗ a

Biotechnology and Molecular Genetics laboratory, Department of Botany, Osmania University, Hyderabad 500007, India Arts and Science College for Women, Andhra Mahila Sabha, OU Campus, Hyderabad, India c College of Science and Arts, Jizan University, Ad Darb, Jizan, Saudi Arabia b

a r t i c l e

i n f o

Article history: Received 30 July 2016 Received in revised form 24 January 2017 Accepted 5 February 2017 Available online xxx Keywords: Commiphora wightii Gum guggul-resin HPLC Guggulsterones Micropropagation

a b s t r a c t Commiphora wightii is an endangered tree valued for its gum guggul-resin that has cholesterol-reducing activity is well documented in alternative systems of medicine. Phytochemical investigations were taken up in six accessions of C. wightii and micropropagation protocol developed for its conservation. The leaves and gum of C. wightii were qualitatively and quantitatively tested for secondary metabolites and an elite accession with very high contents of total sterols and phenols was identified. An efficient HPLC method was utilized in the present study to estimate the valuable and pharmaceutically important steroid compounds present in its gum, viz. E-Guggulsterone and Z-Guggulsterone, which are hypolipidemic agents. The estimation resulted in the values of 2.45 mg/L and 2.17 mg/L for E- and Z-guggulsterone respectively. A highly efficient micropropagation protocol with good rooting and a high plantlet survival was developed from the nodal explants to aid its conservation and 360 plants survived out of 396 plants (transferred to the field) with a high percentage of survival (92.8%). The micropropagation efficiency reported presently in C. wightii far exceeds all the earlier reports and was mainly achieved due to strong rooting and healthier state of plantlets. © 2017 Elsevier GmbH. All rights reserved.

1. Introduction Commiphora wightii (Arnold) Bhandari (known as guggul, guggal, gum guggulu, and gugulipid) is considered endangered in India and is listed in the IUCN Red Data list (IUCN, 2010). The United Nations Development Programme has listed this species as “Critically endangered” in 2008. The Government of India has banned its export and also other over-exploited species (http://www. iucnredlist.org/details/31231/0) in the country (Billore, 1989). C. wightii belongs to family Burseraceae. It is a slow growing high branching shrub that grows to a height of 2–3 m with silvery and paper like gray-brown bark peeling off in small pieces (Barve and Mehta, 1993). The stem is thorny with small leaves. The leaves are alternate or fascicled small, sessile, rhomboid-(ob)ovate, compound, 1–3 (or more)-foliolate, imparipinnate; leaflets sessile or subsessile, serrate, crenate, or entire, highly aromatic, leathery,

∗ Corresponding author. E-mail address: [email protected] (P.D. Cherku).

shinning green on top and greyish below. The plant has poor seed set and germination and has been subjected to indiscriminate and harsh methods of gum-harvesting leading to tree death (Soni, 2010). The oleo gum (resin) is collected by tapping guggul plants in summer and the yield is about 200–800 g per plant. The composition of Guggul is 61% resin, 29.3% gum, 0.6% volatile oils, 6.1% moisture, and 3.2% foreign matter (The Ayurvedic Pharmacopoeia of India, 2001). The guggulosterones present in the guggul gum of C. wightii plants have antioxidant activity (Bhati, 1950). The diuretic activity of isolated fractions from the leaf petroleum ether extract of Commiphora berryi on healthy albino rats was studied with frusemide as reference drug (Selvamani et al., 2005). The leaf and stem extracts of different species of Commiphora were tested positive for the anti-oxidant (ABTS and DPPH assays), antimicrobial (MIC and death kinetic assays), anti-inflammatory (5-LOX assay), anticancer (SRB assay) properties, as well as the cytotoxic effects (tetrazolium-based assay) (Paraskeva et al., 2008) and hence, the leaves must be analyzed for medicinal compounds. The Guggulipids from the resin have been reported to be effective as anti-inflammatory, anti-bacterial, antimicrobial, anti-oxidant,

http://dx.doi.org/10.1016/j.jarmap.2017.02.004 2214-7861/© 2017 Elsevier GmbH. All rights reserved.

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anti-arthritic, anti-malarial, antimycobacterial, anti-schistomal, hepatoprotective, muscle relaxant, larvicidal, and molluscicidal (Atal et al., 1975; Satyavati, 1988; Satyavati, 1990; Sahni et al., 2005; Saxena et al., 2007; Pradhan and Dash, 2011). Studies have reported the cardiac and neuronal protective activity of the steroid guggulsterone (Kaul and Kapoor, 1989).It was found to be beneficial in diabetes (Lather et al., 2011) and also for prevention of cancer (Ramawat et al., 2008; Urizar et al., 2002). Extracts from C. wightii (guggal) are popular in Asia as cholesterol lowering agents and are gaining popularity in the United States (Jachak and Saklani, 2007). Since the plant is very difficult to grow and is also slow growing, there is a need to develop alternative conservation methods like micropropagation for effective production of planting material. Very few reports are available on plant regeneration from cotyledonary node segments (Kant et al., 2010), shoot tip and nodal explants (Barve and Mehta, 1993) and stem cuttings (Soni, 2010). Somatic embryos were reported by Kumar et al. (2004, 2006) from immature zygotic embryo and leaf explants. In vitro regeneration of C. wightii and the influence of growth regulators was reported by Tejovathi et al. (2011). However, more efforts are still needed to establish efficient in vitro regeneration of this endangered medicinal plant. The present study was carried out to investigate the phytochemical characteristics of six accessions of C. wightii with a view to identify the elites in terms of the quality of secondary metabolites, estimate the total steroids and to utilize an efficient HPLC method for the quantitative determination of the E- and Zguggulsterones in the gum exudate of the identified elite accession (s). Further, the study included the development of an efficient micropropagation protocol of C. wightii to aid in its conservation. 2. Materials and methods Six accessions of Commiphora wightii, growing in the Botanical garden, at Osmania University were taken up for the study. These 8 year old accessions were originally sourced from different places of south India, Cw-P (Herbal garden, Rajendranagar, Hyderabad, TS); Cw-R (Karthikavanam Dhulapally, Hyderabad, TS); Cw-S (Hyderabad Urban Forestry, Erragadda, Hyderabad, TS); Cw-U (Andhra Pradesh Medicinal Plants Board, Chilkur, Hyderabad, TS); Cw-Q (Pragathi Green Nursery, Proddutur, AP) and Cw-T (Herbal garden, Rajahmundry, AP) and authenticated by the Head, Department of Botany, Osmania University, Hyderabad. The leaves and gum exudates of the six accessions of C. wightii were screened using qualitative, quantitative and HPLC methods for various phytochemicals of immense medicinal value, especially the E- and Z-guggulsterones with a view to identify an elite which was subsequently multiplied in vitro by the development of a micropropagation protocol for conservation of this valuable, endangered medicinal plant. 2.1. Phytochemical analysis 2.1.1. Preparation of extracts The extracts were prepared from healthy leaves and gum of the all the accessions of C. wightii. Leaf samples were collected, washed under tap water and dried in shade. They were powdered and stored in bottles for future use. The gum was collected from all the accessions by nicking the stem carefully without injuring the phloem. The resin canals are outer to the phloem and therefore deep cuts are not needed. The gum was allowed to dry for a week and then washed thoroughly under running tap water for removal of soil particles and shade dried for about 10 days. After complete drying of the gum, it was ground into a fine powder by passing through a sieve after each grinding (Fig. 1). The samples was kept in air tight containers and protected from light until used. The leaf/gum extract

was prepared (by grinding 0.5 gm of leaf/gum powder in 100 mL of water) and filtered through a muslin cloth before subjecting them to the phytochemical analysis. 2.1.2. Qualitative phytochemical analysis Qualitative tests were carried out for the presence of Alkaloids, Flavonoids, Steroids, Terpenoids, Tannins, Saponins, Glycosides, Phenols and Carbohydrates in the leaves and gum of C. wightii using standard procedures to identify the constituents present in these extracts. 2.1.2.1. Test for Alkaloids. The leaf/gum extract (5 mL) was dissolved in 5 mL dilute HCl solution and filtered. The filtrate was tested with Dragendroff’s and Mayer’s reagent. The treated solution was observed for precipitation. 2.1.2.2. Test for Flavonoids. 5 mL ethyl acetate was added to 10 mL of leaf/gum extract, the mixture was shaken and allowed to settle. Production of greenish yellow color is taken as positive for Flavonoids. 2.1.2.3. Test for Terpenoids. Presence of Steroids and terpenoids in the leaf/gum extract was tested by a mixture of acetic anhydride and chloroform (5 mL each) in presence of concentrated sulphuric acid (2 mL). Appearance of a blue-green ring at the interface between the two liquids indicated the terpenoids. 2.1.2.4. Test for Sterols. 10 gm of leaf/gum powder was added to 10 mL of Liberman-Burchard reagent (0.5 mL of sulphuric acid dissolved in 10 mL of acetic anhydride and stored covered in an ice bucket). The development of a characteristic green color confirmed the presence of sterols. 2.1.2.5. Test for Tannins. To 100 mL of the leaf/gum extract, 10% ferric chloride solution was added and was observed for a change in color to blue. 2.1.2.6. Test for Saponins. Leaf/gum powder (0.5 g) was ground with 100 mL of distilled water and transferred to a test tube. The test tube was shaken vigorously for about 30 s and allowed to stand in vertical position and observed for 30 min. If a honey comb froth above the surface of the liquid persists after 30 min, it indicates the presence of saponins. 2.1.2.7. Test for Glycosides. Presence of Glycosides was tested by adding 5 mL dilute sulphuric acid to 10 mL leaf/gum extract. It was boiled for 5 min, filtered and cooled. Equal volume of chloroform was added and shaken well to separate into two layers. The lower chloroform layer was collected and half volume of ammonia solution added to it which turns pink due to the presence of glycosides. 2.1.2.8. Test for Phenols. The leaf/gum extract (2 mL) was taken in a test tube and warmed. To this, 2 mL of 1% ferric chloride was added and observed for formation of green or blue color. 2.1.2.9. Tests for carbohydrates. Three tests were carried out. 2.1.2.10. Molisch’s test. Presence of carbohydrate was indicated when 5 mL of the extract was slowly mixed with 5 mL Molisch reagent, and later, a small amount of concentrated sulphuric acid was added slowly, leading to the formation of purple ring at the interface.

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Fig. 1. C. wightii gum samples used for phytochemical studies.

2.1.2.11. Benedict’s test. The extract (5 mL) was mixed with 2 mL of Benedict’s reagent, boiled, and observed for the formation of reddish brown precipitate, which indicates the presence of the carbohydrates. 2.1.2.12. Test for reducing sugar by Fehling’s test. Fehling A and Fehling B reagents were mixed together in equal volumes and 2 mL of it was added to 5 mL extract and boiled in water bath and cooled. The appearance of brick red precipitate at the bottom of the test tube indicated the presence of reducing sugars. 2.1.3. Quantitative phytochemical analysis Total sterols and total phenols were estimated. 2.1.3.1. Estimation of total sterols. Estimation of total sterols in the leaf and gum extracts of C. wightii was carried out by the method of Sabir et al. (2003). Fifty gm of leaf/gum powder was dissolved in 200 mL Acetonitrile and incubated at 50–60 ◦ C for 2 h. It was filtered through Whatman No-1 filter paper and 200 mL of methanol was added to it. Three mL of the extract was taken in a test tube with three replicates, and one blank. Two mL of Liberman-Burchard reagent was added to each tube. The reference standard solution was prepared by dissolving 10 gm of standard cholesterol in 10 mL chloroform. This was taken in five test tubes (1, 1.5, 2, 2.5 and 3.0 mL), and chloroform was used as blank. Two mL of Liberman-Burchard reagent was added to each of them to develop the characteristic green color as an indication of sterols. The final volume was made up to 10 mL by adding methanol and incubated in the dark for 15 min. The optical densities of the standard solutions were determined with a spectrophotometer (Elico Ltd.) at 640 nm and the standard graph plotted. The amount of sterols in the leaf/gum sample was determined from the standard graph as mg/gm. 2.1.3.2. Estimation of total Phenols. The total Phenolic content was estimated using Folin-Ciocalteau reagent with a slight modification (Singleton et al., 1999). A quantity of 0.5 mL of the leaf extract was mixed with 1.5 mL (1:10 v/v diluted with distilled water) FolinCiocalteau reagent and allowed to stand for 5 min at 22 ◦ C, after which, 2 mL of sodium carbonate (7% Na2 CO3 , w/v) was added. This was incubated for 90 min in the dark with intermittent shaking until a blue color develops. The absorbance was recorded at 725 nm using a spectrophotometer (Elico Ltd.). The standard graph was prepared by using Gallic acid. The total phenolic content in the

a: Freshly obtained gum from C. wightii.

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b: Dried gum of C. wightii.

leaf extract was expressed as milligrams of Gallic acid equivalent per gram of dry weight (mg GAE/g) of extract. 2.1.4. High performance liquid chromatography (HPLC) studies Chemical analysis provides data to guide improved expression of secondary metabolites. At present the best chemical analysis that depends on initial separation of natural product extracts is High Performance Liquid Chromatography (HPLC) (Grabley and Thiericke, 1998; Julian, 1998). In the present study, the specific accession of C. wightii which recorded the highest quantity of Steroids was further analyzed by HPLC for estimating the concentration of the specific Steroids, EGuggulsterone and Z-Guggulsterone in the gum with the help of the reference compounds, which were used as external standards to set up and calculate appropriate calibration curves. 2.1.4.1. Preparation of gum extracts and sample. Fifty gm of gum powder was dissolved in 200 mL of Acetonitrile (solvent) in 1:4 ratio and incubated at 50 ◦ C–60 ◦ C for 2 h and filtered through Whatman No-1 filter paper. To the filtrate, 200 mL of methanol was added and incubated it at 50 ◦ C–60 ◦ C for 2 h and allowed to dry. The dried extract powder of the gum was weighed accurately (100 mg), dissolved in Acetonitrile in a 25 mL volumetric flask, the final volume made up to the mark and injected (20 ␮L) into the HPLC apparatus. 2.1.4.2. Preparation of the reference standards of E- and Zguggulsterone. The reference standards of E- and Z-guggulsterone were procured from Sigma Aldrich. The stock solutions of the standards were prepared by dissolving accurately weighed 5 mg in Acetonitrile in a 50 mL volumetric flask and made up the volume up to mark. The injected amount was 20 ␮L. 2.1.4.3. HPLC analysis. The experimental conditions were performed using Shimadzu HPLC (LC – 2010 model) (dual wave length) and LC solutions software. A Zorbex SB C-18 (150 × 4.6 mm) 5 ␮m column was used, 0.1% v /v Ortho phosphoric acid in water as eluentA and Acetonitrile-water (0.1-100) as eluent-B. The flow rate of 1 mL/min, Me OH as diluent, at a ␭ max of 243 nm. The weight of standard was 5 mg/mL and the amount of injection was 20 ␮L. The system was gradient with a column temperature of 25◦ C to 30 ◦ C (room temperature). The quantity of E- and Z-guggulsterone was determined from the standard curve. The percentage of compounds present in crude

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guggul-gum extract of C. wightii was calculated using the following formula and expressed as a percentage: Peak area of sample concentration of standard × Peak area of standard concentration of sample ×purity of standard. The retention time and peak area of the respective chromatogram were taken into consideration for quantification of Eand Z-guggulsterone. Identification of compounds was done by comparing the peak of the specific compound in the chromatogram with that of the respective standard peak. 2.2. Micropropagation studies Micropropagation protocol was developed with the nodal explants of the identified elite accession of C. wightii, which was selected based on the qualitative and quantitative studies. Initially, the explants were thoroughly washed under running tap water for about 20 min and transferred to a flask with double distilled autoclaved water containing few drops of Tween-20 with intermittent shaking. They were rinsed thrice with double distilled autoclaved water and treated with 0.1% (w/v) mercuric chloride for 2–4 min. They were rinsed thrice with double distilled autoclaved water, washed with 5% sodium hypochlorite and finally rinsed thrice with double distilled autoclaved water before inoculating them on to sterile culture tubes (25 × 150 mm) containing culture medium on the laminar airflow bench. The Murashige and Skoog (MS) medium (Murashige and Skoog, 1962) supplemented with 3% (w/v) sucrose, solidified with 0.8% (w/v) phyta-agar (Hi-media, India), with various combinations of growth regulators: 6-benzylaminopurine (BAP) either individually or in combination with Naphthalene acetic acid (NAA) and Gibberellic acid (GA3 ), or with Adenine sulphate (AS) and Gibberellic acid (GA3 ) were used. • • • • • • •

MS + BAP (4.44 ␮M) MS + BAP (8.88 ␮M) MS + BAP (4.44 ␮M) + NAA (5.37 ␮M) MS + BAP (8.88 ␮M) + NAA (5.37 ␮M) MS + BAP (4.44 ␮M) + NAA (5.37 ␮M) + GA3 (1.44 ␮M) MS + BAP (2.22 ␮M) + AS (2.47 ␮M) MS + BAP (2.22 ␮M) + AS (2.47 ␮M) + GA3 (1.44 ␮M)

The pH of the medium was adjusted to 5.8 and autoclaved at 121◦ C for 15 lbs./cm 2 for 15 min. Thirty explants were inoculated on each culture medium with three replicates amounting to a total of ninety. The cultures were incubated in a sterile growth room at 25 ± 1 ◦ C with cool fluorescent light (irradiance 80 ␮mol m−2 s−1 ) at 16 h photoperiod with 50–60% humidity. The regenerated shoots (multiple nodes excised into single units) were sub cultured every 3–4 weeks on the same media for elongation. The cultures were evaluated to find the most suitable media. The regenerated shoots (3–4 cm) were transferred to the optimized root induction medium [MS medium supplemented with different concentrations of Indole butyric acid (IBA) were experimented with and the most suitable medium was standardized]. The frequency of regeneration of shoots on the shoot induction media was determined as the percentage of the total cultured explants that responded. The frequency of rooting from the regenerated shoots was determined as the percentage of the total cultured shoots that responded by producing roots. Regenerated plantlets with well-established roots were taken out of the culture tubes carefully, washed with water and transferred to pots containing sand and manure (1:1) and were kept covered with plastic bags

under controlled conditions of 70–80% humidity and temperature of 25 ± 1 ◦ C for about 10-15 days. The plantlets were later transferred to the glasshouse and slowly exposed to outer environment of 32 ± 2 ◦ C with 70–80% relative humidity. Finally the plants were transferred to field after four weeks. The survival percentage of plantlets transferred to the field was calculated after three months and presented as the percentage of established plants out of the total rooted plantlets that were transplanted. 2.3. Statistical analysis All the experiments of phytochemical analysis and micropropagation were set up in a completely randomized design with three replications per treatment and the assays were performed in triplicate to verify the reproducibility of the results and expressed as mean ± SE. 3. Results and discussion The present report concerns the qualitative and quantitative phytochemical analysis of leaf and gum extracts of six accessions of C. wightii (Cw-P), (Cw-Q), (Cw-R), (Cw-S), (Cw-T) and (Cw-U), followed by the HPLC analysis of leaves and gum of C. wightii for quantification of Steroids (E-guggulsterone and Z-guggulsterone) and the development of a micropropagation protocol from the nodal explants of the identified elite accession. 3.1. Phytochemical analysis 3.1.1. Qualitative analysis Qualitative phytochemical analysis carried out on the leaf and gum extracts of C. wightii have shown the presence of secondary metabolites and sterols (Table 1). The leaves of all the accessions of C. wightii contained Flavonoids, Steroids, Terpenoids, Tannins, Phenols and Carbohydrates. Alkaloids are absent in the leaves of all the accessions. Saponins and Glycosides are also absent except in one accession. The gum exudate of all accessions contained only Steroids, Terpenoids and Carbohydrates. These results were similar to the report of Rani and Mishra (2013). It has been confirmed that phenolic compounds like tannins present in the cells of plants are potent inhibitors of many hydrolytic enzymes such as proteolytic macerating enzymes used by plant pathogens (Cowan, 1999). Therefore, a very high phenolic content is hence desirable. The use of plants for medicinal purpose is in existence since ancient times and they were subjected to the extraction and development of several drugs apart from traditionally being used as folk medicine (Shrikumar and Ravi, 2007). The secondary metabolites in the plants play important role in human health and are nutritionally important (Hertog et al., 1993). Sometimes the crude extracts of medicinal plants are more active biologically than isolated compounds due to their synergistic effects (Jana and Shekhawat, 2010). Secondary metabolites of plants plays an important role in defense mechanism against predation by many microorganisms, insects and herbivores (Cowan, 1999). 3.1.2. Quantitative analysis Quantitative phytochemical analysis (for Steroids) of the leaf and gum extracts was carried out in six accessions of C. wightii for Steroids and analysis of the leaf extracts was carried out in six accessions of C. wightii for Phenols. In the leaf extracts, the highest amount of Steroids (0.18 ± 0.05 mg/100gm) was recorded in the Cw-P accession of C. wightii and in the gum also the highest Steroid content (0.48 ± 0.05 mg/100gm) was recorded in the Cw-P accession (Table 2). The highest content of Phenols (1.434 ± 0.09 mg GAE/g) was recorded in the leaf extract of Cw-P (Table 3). Therefore, among all the accessions of C. wightii, the Cw-P accession has

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Table 1 Qualitative phytochemical analysis of 6 accessions of C. wightii. S No

Phytochemicals

Leaf extracts of accessions Cw-P

Cw-Q

Cw-R

Cw-S

Cw-T

Cw-U

1 2 3 4 5 6 7 8 9

Alkaloids Flavonoids Steroids Tannins Terpenoids Saponins Glycosides Phenols Carbohydrates

− + + + + − − + +

− + + + + − − + +

− + + + + − − + +

− + + + + − − + +

− + + + + + + + +

− + + + + − − + +

S. No

Phytochemicals

Test results of gum extracts of accessions Cw-P

Cw-Q

Cw-R

Cw-S

Cw-T

Cw-U

1 2 3 4 5 6 7 8 9

Alkaloids Flavonoids Steroids Tannins Terpenoids Saponins Glycosides Phenols Carbohydrates

− − + − + − − − +

− − + − + − − − +

− − + − + − − − +

− − + − + − − − +

− − + − + − − − +

− − + − + − − − +

+ Presence of the compound. − Absence of the compound. Table 2 Quantitative estimation of sterols in leaves and gum of 6 accessions of C. wightii. Accessions

Content of sterols in leaf extract (mg/100 gma )

Content of sterols in gum extract (mg/100 gma )

Cw – P Cw – Q Cw – R Cw – S Cw – T Cw – U

0.18 ± 0.05 0.05 ± 0.08 0.06 ± 0.02 0.05 ± 0.04 0.03 ± 0.01 0.03 ± 0.06

0.48 ± 0.05 0.35 ± 0.08 0.36 ± 0.02 0.25 ± 0.04 0.33 ± 0.03 0.34 ± 0.06

a

Per 100 gm of leaf/gum powder.

Table 3 Total phenolic content in leaf extracts of six accessions of C. wightii. The analysis was carried out in triplicate and expressed as Mean ± SE

b

a b

S. No

Accessions

Phenolic content in leaf extract (mg GAE/ga ) (Mean ± SE)b

1 2 3 4 5 6

Cw – P Cw – Q Cw – R Cw – S Cw – T Cw – U

1.434 ± 0.09 0.253 ± 0.08 0.696 ± 0.06 1.046 ± 0.05 0.568 ± 0.04 0.434 ± 0.09

Milligrams of gallic acid equivalent per gram of dry weight (mg GAE/g) of extract. The analysis was carried out in triplicate and expressed as Mean ± SE

the highest concentration of Steroids in the leaves and gum compared to other accessions. The Steroid content was higher than those reported earlier (Agrawal et al., 2004; Verma et al., 1998). Although the gum is a storehouse of valuable compounds as seen here as well as the previous reports, the leaves also have a lot of potential in possessing medicinal compounds which needs to be explored further (Selvamani et al., 2005; Paraskeva et al., 2008). Due to its highest Steroid content, the Cw-P accession was used for estimation of E-guggulsterone and Z-guggulsterone by HPLC analysis.

3.1.3. HPLC analysis of gum extract In the present study, the identified elite accession of C. wightii (Cw-P) was screened for two secondary metabolites E-Guggulsterone and Z-Guggulsterone (sterols) by High performance Liquid Chromatography (HPLC) method. This accession was selected because, it showed the highest percentage of Steroids in quantitative phytochemical analysis. Ayurvedic physicians extensively used guggul gum for treating arthritis and related conditions for centuries. Two biologically active components E-guggulsterone and Z-guggulsterone were identified in Commiphora mukul crude resin extracts (Bohos et al., 1998). In the present study, HPLC analysis was carried out for estimation of the two important secondary metabolites (Eguggulsterone and Z-guggulsterone) from the gum of an accession of C. wightii (Cw-P). HPLC method was preferred as analytic tool for quantification of the compounds in C. wightii because of its popularity in analysis of herbal medicine, accuracy, precise, simplicity, sensitivity and it is not limited by the volatility of stability of the sample compounds (Klejdus et al., 2001; Jarkko and Pirjo, 2008). The stationary phase for quantification of the secondary metabolites (E-guggulsterone and Z-guggulsterone) was the symmetry shield (Zorbex SB C-18 (150 × 4.6 mm) with two mobile phases (mobile phase A-0.1% v /v Ortho phosphoric acid in water, B- Acetonitrile-water; (0.1–100), methanol). Throughout the run the flow rate was maintained at 1 mL/1 min. The column effluent was monitored at 243 nm with 25 ◦ C–30 ◦ C temperature. Initially, the standards of E-Guggulsterone and Z-Guggulsterone were run individually with the standardized parameters (given above). Later, the gum extract of C. wightii (accession Cw-P) was run and the identification of compounds was done by comparing the peak of the specific compound in the chromatogram with that of the respective standard peak. The quantity of E-Guggulsterone and ZGuggulsterone from the gum extract of C. wightii was determined from the respective standard curve.

3.1.3.1. Chromatograms of the standards. The retention time for standard E-Guggulsterone was recorded with three runs in HPLC and was observed as 9.306 min at 243 nm (Fig. 2). The retention

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Fig. 2. HPLC graph showing the standard E-Guggulsterone.

time for standard Z-Guggulsterone was recorded with three runs in HPLC and was observed as 11.531 min at 243 nm (Fig. 3).

The estimated concentration of E- and Z-guggulsterones and the trend were similar to those of Zhu et al. (2001), Verma et al. (1998) and Bohos et al. (1998).

3.1.3.2. Chromatogram of the gum extract of C. wightii. For EGuggulsterone: The HPLC chromatogram of the gum extract showed a retention time of 9.279 min at 243 nm for EGuggulsterone (Fig. 4). After running the HPLC, the standard and gum extracts showed different retention times. To detect the compound exactly from the chromatogram peak, both the samples (50% + 50% of the standard and gum extract respectively) were run twice in HPLC and then, by spiking the highest peak in the chromatogram, the compound was identified as E-Guggulsterone. The concentration of E-Guggulsterone in the gum extract of C. wightii was detected as 2.45 mg/L. For Z-Guggulsterone: The HPLC chromatogram of the gum extract showed a retention time of 11.495 min at 243 nm for ZGuggulsterone (Fig. 4). After running the HPLC, the standard and gum extracts showed different retention times. To detect the compound exactly from the chromatogram peak, both the samples (50% + 50% of the standard and gum extract respectively) were run twice in HPLC and then, by spiking the highest peak in the chromatogram, the compound was identified as Z-Guggulsterone. The concentration of Z-Guggulsterone in the gum extract of C. wightii was detected as 2.17 mg/L.

3.2. Micropropagation The present study comprises experiments conducted for the selection of appropriate culture media for carrying out micropropagation of C. wightii. It reports a rapid, reliable and reproducible protocol employing the nodal explant for efficient micropropagation. In vitro culture was carried out with nodal explants of the 8-year-old accession Cw-P of C. wightii because it was identified as an elite accession possessing significantly high amounts of medicinal components. Explants were collected from the tree growing in the Botanical garden, Department of Botany, Osmania University. In vitro propagation is an effective means of rapid multiplication of species in which conventional methods have limitations (Nehra and Kartha, 1994). In vitro propagation is a powerful tool for germplasm conservation and the mass multiplication of threatened plant species (Murch et al., 2000). Micropropagation is an excellent technique that uses plant tissue culture method to obtain maximum number of regenerates within a short period. Although there are a few recent reports on direct plant regeneration of C. wightii by Soni (2010), Tejovathi et al. (2011) and Parmar and Kant (2012) from different explants, the efficiency needs improvement.

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Fig. 3. HPLC graph showing the standard of Z-Guggulsterone.

Table 4 Response of nodal explants of the Cw-P accession of C. wightii in production of shoots. S. No

Culture medium

a

1 2. 3. 4. 5. 6. 7.

MS + BAP(4.44 ␮M) MS + BAP(8.88 ␮M) MS + BAP(4.44 ␮M) + NAA(5.37 ␮M) MS + BAP(8.88 ␮M) + NAA(5.37 ␮M) MS + BAP(4.44 ␮M) + NAA(5.37 ␮M) + GA3 (1.44 ␮M) MS+ BAP(2.22 ␮M) + AS (2.47 ␮M) MS + BAP(2.22 ␮M) + AS(2.47 ␮M) + GA3 (1.44 ␮M)

30 × 3 30 × 3 30 × 3 30 × 3 30 × 3 30 × 3 30 × 3

a

Explants inoculated

Response (%)

Frequency of shoots (Mean ± S.E.)a

40 46 53 50 60 83 90

12.00 ± 0.60 13.80 ± 0.32 16.80 ± 0.56 15.00 ± 0.52 18.00 ± 0.42 24.90 ± 0.42 27.00 ± 0.52

All the analysis were carried out in triplicate and expressed as mean ± SE.

In the present study, various combinations and concentrations of growth regulators have been used as supplementation to MS medium to develop a highly efficient micropropagation protocol from nodal explants. The explants were cultured on seven different culture media based on the MS medium but differing with each other in terms of the growth regulators. The explants of C. wightii cultured on different culture media responded in different ways. The highest response (90%) of shoot regeneration was observed on MS + BAP (2.22 ␮M) + AS (2.47 ␮M) + GA3 (1.44 ␮M) medium (Table 4). Although BAP individually could elicit moderate response, the addition of NAA helped in the increase of shoot production (Figs. 5a and 5b). Hence, the use of BAP along with AS + GA3 further helped in increasing the effi-

ciency of shoot production. Therefore, of all the culture media used for micropropagation of C. wightii, the highest efficient response of shoots was recorded with the culture medium MS + BAP (2.22 ␮M) + AS (2.47 ␮M) + GA3 (1.44 ␮M). Of both MS + BAP + NAA and MS + BAP + AS combinations (with or without GA3 ), the most efficient was MS + BAP + AS in the present study which is in contrast to the earlier reports which have used other than the above combinations: MS + BAP + NAA: (Parmar and Kant, 2012); MS + kn + BAP + IBA (Soni, 2010); MS + KN + NAA + IBA + GA3 (Tejovathi et al., 2011). The explants of C. wightii with regenerated shoots were selected, shoots excised and transferred to rooting medium (Fig. 5c). For root induction, preliminary studies were carried out with differ-

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Fig. 4. HPLC graph showing the analysis of the gum extract of the accession Cw-P for E- and Z-Guggulsterones.

Table 5 Rooting and establishment of regenerated plants of the Cw-P accession of C. wightii. S. No

Shoot regeneration medium

No. of shoots transferred to rooting medium (MS + 9.84 ␮M IBA)

No. of rooted shoots

Rooting response (%)

Survival of plantlets in the field (%)

No. of established plants in the fielda

1 2 3

MS + BAP MS + BAP + NAA MS + BAP+ NAA + GA3 MS + BAP +AS

96 112 111

80 90 100

83.30 80.35 90.09

87.50 91.11 91.00

70 82 91

130

126

96.90

92.85

117

4 a

90.90% of the transplanted plants survived in the field.

ent media comprising of MS medium with IAA or IBA. The most suitable root induction medium that induced highest number of roots was MS+ IBA (9.84 ␮M) (Table 5) (Fig. 5d). The rooting ranged from 83 to 96%. In contrast to the present results, Parmar and Kant (2012) reported the best rooting on MS + BAP (8.88 ␮M) + IAA (5.71 ␮M). Regenerated plantlets with well- developed roots were acclimatized by transferring the plantlets to pots containing sand and manure (1:1) and were kept covered with plastic bags under controlled conditions (25 ± 1 ◦ C) for about 10–15 days (Fig. 5e). The plantlets were initially acclimatized in pots covered by plastic bags to retain high humidity in controlled conditions (25 ± 1 ◦ C) and slowly exposed to outer environment. The pots were then transferred to the glasshouse (Fig. 5f). The temperature of the glasshouse was maintained at 32 ± 2◦ C and 70–80% relative humidity. The sur-

vival percentage of plants in the field ranged from 87 to 92% (360 plants survived out of 396 plants transferred to the field) (Table 5). However, the micropropagation efficiency reported presently in C. wightii far exceeds all the earlier reports and 360 plants survived out of 396 plants transferred to the field with a much higher percentage of survival of plantlets in the field (92.8%) mainly achieved due to strong rooting and healthier state of plantlets. The protocol developed in the present study for shoot regeneration and rooting would be useful in their micropropagation and can help conserve the endangered plants. 4. Conclusion The qualitative and quantitative analysis of plant parts of C. wightii for screening and estimation of various secondary metabo-

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Fig. 5. Micropropagation of C. wightii (accession Cw-P) from nodal explant. a. The nodal explant 1 week after inoculation on MS Medium + BAP + AS + GA3. b. Elongated shoots from nodal explants after 4 weeks of inoculation. c. Intiation of roots from the regenerated shoots of nodal explant of C. wightii. d. Regenerated plantlet with well developed roots after 13 weeks of growth. e. Acclimatization of the regenerated C. wightii plantlet in glasshouse in controlled conditions. f. Acclimatized C. wightii plantlet.

lites is completely described in detail for easy replication. It provides valuable information to the pharmaceutical industry for preparation of drugs. The successful HPLC technique developed for quantitation has high sensitivity. All the methods adopted presently for the analysis of two biologically active components

E- and Z-guggulsterones are selective, accurate and reproducible and an elite accession has been identified with high contents of the Steroids. The protocols developed in the present study for high frequency shoot regeneration and rooting would be useful in their

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