Keywords

8.1 Introduction

The trans-Himalayan regions are very exceptional in the world with extreme temperature variation, high ultraviolet radiation, and low availability of oxygen which are difficult for human survival and results in cognitive dysfunctions, high altitude region maladies, and lowered performance due to alteration in biological functions. However, the Himalaya region is also a reservoir of diverse flora and fauna and to fight and service with these problems in these difficult situations. Plants found in high region above 3000 m of Himalayas are extensively used in ethnomedicine/traditional medicinal system, both as prophylactics remedy and therapeutics. The valuable effects of these herbal drugs/supplements in combating the adverse effects of high altitude region have been deeply investigated in recent years. The plant species is important due to the efficient medicinal properties with slightest or no side effects.

Rhodiola imbricata Edgew. species known to contain the active chemical compounds of the class flavonoid glycosides and coumarins (Khanum et al. 2005). Phenylpropanoids and phenylethanol derivatives are the major compounds from these Rhodiola plants, particularly roots or rhizomes, which have been shown to have various biological activities (Kelly 2001; Yousef et al. 2006). Anti-inflammatory, cardioprotective, antipyretic, anti-stress, and adaptogenic effects are the major pharmacological activities exhibited by R. imbricata (Chawla et al. 2010; Gupta et al. 2010; Mishra et al. 2012; Tayade et al. 2013a, b, c). The rhizomes exhibits wound healing (Gupta et al. 2007), radioprotective (Arora et al. 2005), antioxidant, cytoprotective (Kanupriya et al. 2005), adaptogen (Gupta et al. 2008; Spasov et al. 2000), immunomodulatory (Mishra et al. 2006), neuroprotective (Mook-Jung et al. 2002), and antiproliferative activities (Senthilkumar et al. 2013).

8.2 Morphology and Distribution of Golden Root

Rhodiola imbricata (Synonyms: Sedum imbricatum (Edgew.) Walp.) is a high altitude perennial herb of Crassulaceae family, commonly known by different names such as rose root, arctic root, golden root, Himalayan stone crop, shrolo marpo, and lalphool. There are about 200 species of Rhodiola distributed worldwide. This species grows usually on rocky substratum on slopes and also prefers sandy area at high altitudes of Asia and Europe (Chaurasia and Singh 1996). In India, R. imbricata is mainly distributed in the Trans-Himalayan Cold Desert at the altitude ranges between 4000 and 5000 m above sea level in Leh-Ladakh region in Himalaya (Chaurasia and Singh 1996). A schematic representation of habit, part used, and active marker compounds present in Rhodiola imbricata is given in Fig. 8.1a–c.

Fig. 8.1
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Morphology and chemical markers of Rhodiola imbricata ((a). Habit of R. imbricata in wild, (b). Part used, (c). marker compounds)

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8.3 Traditional Uses

Usually the genus Rhodiola root has been used in folklore medicine to increase physical endurance, longevity, to treat impotence, and gastrointestinal disorders (Kelly 2001). This species is considered as one of the major ingredient of local tea (herbal), acting as antioxidant biological function (Mishra et al. 2008). It is widely used as food as well as medicine for curing common diseases around the world. This plant species is used in Chinese phytotherapy and Amchi system to cure various ailments and helps to maintain body function. In Leh-Ladakh (UT), this species is used as wild edible, fodder, and an ornamental plant. The boiled shoots with water used and mixed with yogurt to make tantur, a local delicious food item.

8.4 Phytochemistry

Several secondary metabolites such as phenylethanol derivatives, phenylpropanoids, phenolic acids and flavonoids were reported from R. imbricata. HPLC analysis of acetone extract established the occurrence of gallic acid and rutin (Senthilkumar et al. 2014)) in the plant. The aqueous extract reported with major bioactive compounds such as rosin, rosavin, and p-tyrosol (Mishra et al. 2008). The GC-MS analysis of methanolic extract (ME) showed phytosterols, phenols, alkyl halide and fatty acid esters (Tayade et al. 2013a, b, c). Rhodiola imbricata possesses phenolic compounds like salidroside and p-tyrosol as primary and secondary metabolites (Guo et al. 2014). Total of 19 compounds have been isolated (Figs. 8.2a and 8.2b) from ethyl acetate and n-butanol extracts through preparative HPLC (Choudhary et al. 2015). Chemometric profile of the n-hexane, CHCl3, CCl4, EtOAc, CH3OH, and 60% C2H5OH root extracts through GC-MS technique revealed the occurrence of 63 phyto-chemotypes (Tayade et al. 2013a, b, c).

Fig. 8.2a
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Chemical constituents of Rhodiola imbricata

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Fig. 8.2b
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Chemical constituents of Rhodiola imbricata

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8.5 Phyto-chemotypes Identified through GC-MS

8.5.1 Phyto-chemotypes Identified from n-Hexane Root Extract

The root extract in n-hexane was identified to contain the major compound as 28.2% 1-pentacosanol; other constituents include, 17-pentatriacontene (7.1), stigmast-5-en- 3-ol, (3β,24S) (13.4), 1-tetracosanol (9.3), 1-henteracontanol (8.5)and 13-tetradecen-1-ol acetate (6.4), 1-heptacosane (3.5), 1-hentriacontane (3.6), 1-tericosanol (2.5), α-tocopherol-β-D-mannoside (0.7), 13-docosan-1-ol, (Z) (2.2), eicosen-1-ol, cis-9 (1.9) stigmast-4-en-3-one (1.3), bis (2-ethylhexyl) phthalate (1.2), hexadecanoic acid (1.2), 1- tetrateracontane (0.9), campesterol (0.9), stigmastanol (0.7), and 3-methoxy-5-methylphenol (0.5). The structures of phyto-chemotypes identified from R. imbricata are given in Figs. 8.3a, 8.3b, 8.3c, 8.3d and 8.3e.

Fig. 8.3a
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Phyto-chemotypes identified through GC-MS from R. imbricata

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Fig. 8.3b
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Phyto-chemotypes identified through GC-MS from R. imbricata

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Fig. 8.3c
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Phyto-chemotypes identified through GC-MS from Rhodiola imbricata

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Fig. 8.3d
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Phyto-chemotypes identified through GC-MS from R. imbricata

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Fig. 8.3e
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Phyto-chemotypes identified through GC-MS from R. imbricata

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8.5.2 Phyto-chemotypes Identified from Chloroform and Dichloromethane Root Extract

In the root extract with chloroform, the chemical compounds characterized were 24.30% stigmasterol, 14.64% methyl tri-butyl NH4Cl, and 11.50% bis (2-ethylhexyl) phthalate; other identified compounds were 7,8-dimethylbenzocyclooctene (7.97), ethyl-linoleate (4.75), 3-methoxy-5-methylphenol (4.16), and hexadecanoic acid (4.13) were found in major amount while campesterol (3.94), benzene methanol, 3-hydroxy-5-methoxy (2.62),17-pentatriacontene (3.38), benzene sulfonic acid, 4-amino-3-nitro (3.21), orcinol (2.93), 1- tetracosanol (1.86), stigmast-4-en-3-one (1.82), α-tocopherol (1.31), and eicosen-1-ol, cis-9 (1.13). In dichloromethane root extract, the most dominant isolates were 17.78% camphor and 15.42% stigmast-5-en-3-ol(3β,24S); other metabolites were mostly identical to chloroform extract in different ration.

8.5.3 Phyto-chemotypes Identified from Ethyl Acetate Root Extract

In ethyl acetate extract, the major isolates were 1,3-dimethoxybenzene and 1,3-benzenediol, 5-pentadecyl which were represented by 27.6 and 16.9%, respectively; other chemical constituents include 1,3-benzenediol, 5-methyl (8.4), 3-methoxy-5-methylphenol (10.1), benzenemethanol, 3-hydroxy, 5-methoxy (5.7), cholest-4-ene-3,6-dione (5.7), dodecanoic acid, 3-hydroxy (4.5), 7,8-dimethylbenzocyclooctene (3.6), 3,5-dimethoxyphenyl acetate (3.4), 1-dodecanol, 3,7,11-trimethyl (0.6), stigmasterol (2.2), hexadecanoic acid (1.8) and oleic acid (1.4).

8.5.4 Phyto-chemotypes Identified from Ethanol Root Extract

In this root extract, the major constituents were dotriacontane and heptadecane, 9-hexyl represented by 5.7 and 5.4%; others in trace amount include hexadecanoic acid, methyl ester (2.3), bis (2-ethylhexyl) phthalate (3.6), and dibutyl phthalate (1.3).

Total of six fat-soluble vitamins (A, E, D2, D3, K1, K2) and nine water-soluble vitamins (B1, B2, 2 number B3, B5, B6, B7, B9, B12) were analyzed from the root of R. imbricata by the RRLC-MS/MS (Tayade et al. 2013a, b, c).

8.6 Biological Aspects

8.6.1 Immunomodulatory Activity

8.6.1.1 IL-6, TNF-α, and NO Production

Aqueous extract (AE) causes increase in TNF-α and IL-6 production in PBMCs (human) and RAW 264.7 cells. AE also causes significant increase in NO- and LPS-induced production when human cells are incubated for 24 h (probability <0.05).

8.6.1.2 Phosphorylated IκB in PBMCs

The expression of p-IκB increased when the cells were exposed with AE at concentration of 250 μg/ml after 40 min, while decreased expression of p-IκB was observed after 60 and 80 mins exposure to the extract.

8.6.1.3 Activation of Transcription Factor NF-κB in PBMCs

Aqueous extract (250 μg/ml) accelerates the nuclear translocation of NF-κB in human PBMCs (Mishra et al. 2006).

8.6.2 Radiomodulatory and Free-Radical Scavenging Activity

8.6.2.1 Electron Donation Ability of Hydro-alcoholic Fractionated Extract

A dosage-dependent increase was observed in electron donation ability of extract as does the absorbance. Higher reducing power was achieved at lower AUV.

8.6.2.2 O•-2 Quenching Ability of Hydro-alcoholic Fractionated Extract

The O•-2 scavenging potential of extract was significantly higher (p < 0.05) between 0.025 and 0.250 mg/ml. Both exhibit >60% activity beyond this concentration (IC50 ≤ 0.025 mg/ml).

8.6.2.3 NO Scavenging Potential of Hydro-alcoholic Fractionated Extract

A dose-dependent increase in NO radical scavenging potential of extract was observed, which was considerably better as compared to standard drug ascorbic acid (vitamin C) at all the tested concentrations. Vitamin C indicated lowering properties at dosages of 0.75 mg/ml. The higher activity was observed at 2 mg/ml (60.38 ± 1.84%) (IC50 = 0.5 mg/ml).

8.6.2.4 Erythrocyte Protection Potential of Hydro-Alcoholic Fractionated Extract

There was 80% hemolysis inhibition with 100 μg/ml. But beyond 500 μg/ml, the extract showed rise of hemolysis upto 2 mg/ml (Arora et al. 2008).

8.6.3 Anti-cellular and Immunomodulatory Activity

8.6.3.1 Hemolytic Activity

Aqueous extract did not show any hemolytic effect on RBCs of human blood samples when tested in various concentrations.

8.6.3.2 Effect on HL60 and EL-4 Cells Proliferation

Proliferation of erythroleukemic HL60 cells was significantly inhibited by the aqueous extracts (100 and 200 μg/ml) in dose-dependent manner.

8.6.3.3 ELISPOT Assay for TNF-α

Enhanced TNF-α spots were observed when human PBMCs were treated with the extract by ELISPOT assay.

8.6.4 Immunopotentiating Activity

Detectable antibody response to tetanus toxoid (TT) and ovalbumin (OVA) was observed with CFA and aqueous extract treated rats. Antibody titers after 27 days of immunization generated were >1:20,000 against TT which was considerably higher than PBS immunized control rats. Antibody response observed with OVA and CFA was detectable up to dilutions of 1:100; however, aqueous extract in combination with OVA had shown superior response at higher dilution (1:5000). The efficacy of aqueous extract was determined by using strong antigen TT and weak antigen OVA. Therefore, this study suggested that aqueous extract had adjuvant/immune potentiating activity in terms of humoral as well as cell-mediated immune response against strong antigen like TT and weak antigen like OVA (Mishra et al. 2010).

8.6.5 Anticancer Activity

8.6.5.1 Effect on Cell Viability

Aqueous extract of 200 μg/ml has no adverse effect on human erythro-leukemic K-562 cells viability in comparison to untreated controls.

8.6.5.2 Effect on Proliferation of K-562 Cells

Dosage-dependent inhibition in erythro-leukemic K-562 cells proliferation was observed when treated with aqueous extract, and decrease in proliferation at 100 and 200 μg/ml was observed.

8.6.5.3 Measurement of ROS in K-562 Cells

A significant increase in reactive oxygen species (ROS) generation was recorded when cells were treated with aqueous extract (200 μg/ml) as compared to control.

8.6.5.4 Effect on Apoptosis

There was a marked increase in apoptosis (17%) in K-562 cells when treated with 200 μg/ml aqueous extract compared to untreated control (11%) with Annexin V FITC and PI after staining.

8.6.5.5 Effect on Cell Cycle

Increase in G2/M phase cells percentage was observed significantly when K-562 cells treated with aqueous extract (200 μg/ml), and aqueous extract arrests cell cycle progression in G2/M phase.

8.6.5.6 Effect on NK Cell Cytotoxicity

About 200 μg/ml aqueous extract of this species significantly observed to increase NK cell cytotoxicity with effector/target ratio of 50:1, and suggested potent anticancer properties useful in treatment of leukemia cancer (Mishra et al. 2008).

8.6.6 Radioprotective Activity

8.6.6.1 Maximum Tolerated Dose (MTD)

Aqueous and aqua-alcoholic extract single dosages administered to mice was well tolerated up to 1.1 and 1.3 gm/kg of body weight, respectively, without any noticeable adverse effect with 3 days. However, beyond these levels, mortality was observed in a dose-dependent manner.

8.6.6.2 Hemopoietic Stem Cells Protection

CFU counts in treated control mice, 7.5Gy have 1.91 ± 0.15. The aqueous extract pretreated irradiated mice had higher CFU (17.3 ± 0.7) than the aqua-alcoholic pretreated irradiated mice (15.6 ± 0.6). The relative increases over untreated irradiated control were 9.1- and 8.3-fold, respectively. The radioprotective effects generated in terms of survival and increased hemopoiesis led to high curative gains which indicated the potential of this species in the development of a radioprotector (Goel et al. 2006). The extract up to 1400 mg/kg BW was well tolerated by mice without any adverse effect, except for the mice being a little drowsy for 3–5 mins, while in higher doses mortality was observed in a dose-dependent manner. Mice exposed to 10Gy gamma-radiations died before 12th post-irradiation day (Arora et al. 2005).

8.6.7 Dermal Wound Healing

1.0% w/v of extract causes significant wound healing when applied topically, 20 μl two times daily for 1 week (Gupta et al. 2007).

8.6.8 Immunological Properties of R. imbricata Aqueous Extract

8.6.8.1 TLR4-MD2 Expression by Flow Cytometry

The splenocytes dosages with aqueous extract of 25 and 50 μg/ml indicated noticeable increase in the expression of TLR4-MD2 compared to the control group.

8.6.8.2 Enhanced Intracellular Granzyme-B Production

The PE-granzyme-B fluorescence intensity normally used to examine the expression of intracellular granzyme-B in splenocytes. Dosages of aqueous extract at 50 μg/ml for 3 days induced a major increase in expression of Granzyme-B, and this indicated helpful in killing of cancerous cells infections.

8.6.8.3 Increased Production of TH1 Cytokines

PBMCs of human used for evaluating the effect of aqueous extract on production of TNF-γ, IL-1β, IL-2, IL-6, IL-8, TNF-α, IL-10, IL-4, and TNF-β. The hPBMCs treating with aqueous extract at 50, 25, and 12.5 μg/ml records increasing IL-2, IL-1β, IL-6, and TNF-α levels (Mishra et al. 2009).

8.6.9 Antiproliferative Activity

It was found that 84% acetone and CH3OH extracts inhibited the proliferation of HT-29 cells treated at 200 μg/ml concentration (Senthilkumar et al. 2013).

8.6.10 Radioprotective Activity

Methanolic fraction exhibited significant antioxidant activity at 250 μg/ml and contributed to radioprotective efficacy. The solvent extraction and dose are essential in bioactivity modulation; this plant species could be developed as a possible prophylactic radiation counter measure for nuclear and radiological emergencies (Chawla et al. 2010).

8.6.11 Safety Study

The higher dosages of 250 and 500 mg/kg extract increase BW of rats (both sexes). Increase in physiological parameters, that is, plasma glucose and protein levels, was recorded at both the dosages, which were observed to be restored to normal after 2 weeks withdrawal of treatment (Tulsawani et al. 2013).

8.6.12 Antioxidant Activity

The reduction capability of DPPH radicals was determined by the decrease in its absorbance at 515 nm induced by antioxidants. The DPPH activity of the plant extract was increased at a dose of 0.1–1.2 mg/ml in a dose-dependent manner, and found between 39.55 and 70.76% as compared to standard ascorbic acid 46.78–81.47%. The inhibitory concentrations (IC50) of methanol extract (ME) in DPPH radical, nitric oxide, and OH radical were observed to be 0.33, 0.47, and 0.58 mg/ml, respectively (Kumar et al. 2010a, b). Total flavonoid and flavonol contents were probable to be 30.2, 17.67, 20.68, and 7.38 mg quercetin equivalent/g of the plant extract, respectively. The ME exhibits higher antioxidant capacity as compared to aqueous extract (Tayade et al. 2013a, b, c). Senthilkumar et al. (2013) recorded methanol (IC50 62.80 μg/mL) and acetone (IC50 63.80 μg/mL) extracts exhibit the maximum DPPH radical scavenging activity, which is similar to the reference standard BHT (IC50 45.56 μg/mL). At different concentrations (100–1000 μg/ml), R. imbricata demonstrated powerful reducing capacity, and root extract records the maximum antioxidant capacities (Kumar et al. 2010a, b).

8.6.12.1 Free Radical Scavenging Activity

Quercetin IC50 value, aqueous extract, and BHT were found to be 3.824, 4.391, and 4.743 μg/ml, respectively, and the extract exhibited significant dose-dependent inhibition.

8.6.12.2 Effect on Anti-Lipid Peroxidation Activity

Aqueous extract at 500 μg/ml concentration exhibits the maximum scavenging activity, while the least scavenging activity was observed at 0.5 μg/ml concentration. The IC50 values of aqueous extract and α-tocopherol were found to be 5.12 and 4.89 μg/ml, and this suggested inhibition of lipid peroxidation due to free radical scavenging properties.

8.6.12.3 Total Phenolic Content

Gallic acid equivalent of phenols present in 1 g of aqueous extract is 240.0 ± 10 mg (Gupta et al. 2009a, b).

8.6.12.4 Hydrogen Peroxide Radical Scavenging Activity

Aqueous extract had effective H2O2 scavenging activity (3.254 μg/ml) as compared to standard (4.004 μg/ml).

8.6.12.5 Total Flavonoid Content

The total flavonoid content was found to be 66.7 μg quercetin equivalent/mg of aqueous extract.

8.6.13 Antioxidative Effect during Cold, Hypoxia, and Restraint Exposure

Antioxidative potential of R. imbricata root aqueous extract (100 mg/kg) was examined in rats. It was administered orally 30 min prior to cold (5 °C)–hypoxia (428 mmHg)–restraint (C–H–R) exposure. There was restricted increase in blood MDA, GSH (glutathione),) and SOD (superoxide dismutase) activity with limited rise in blood, muscle, and liver LDH; improved muscle and liver SOD on attaining Trec23 and Trec37 °C; liver CAT on attaining Trec23 °C and liver GST during recovery when administered with single dose of aqueous extract.

8.6.13.1 Adaptogenic Activity

Studies by Gupta et al. (2008) record 100 mg/kg drug dosages significantly provide the maximum resistance to C–H–R induced hypothermia (103.9%) and speed up the recovery rate by 36.8%. One dose of plant extract considerably increased time required to attain Trec23 °C during C-H-R exposure, and it also decreased time recover (Trec37 °C) after C-H-R exposure as compared with experimental controls (Gupta et al. 2009a, b).

8.6.13.2 Acute Toxicity

There was no mortality when rats were treated at oral doses of 1, 2, 5, and 10 g/kg with aqueous extract.

8.6.14 Cytoprotective Activity

An increase in cytotoxicity and apoptosis was recorded significantly over control cells in tert-BHP presence (Table 8.1). Both aqueous and alcoholic extracts (250 μg/ml) were observed to inhibit tert-BHP induced free radical production and cell death. These results clearly suggested R. imbricata had marked cytoprotective and antioxidant activities (Kanupriya et al. 2005).

Table 8.1 Rhodiola imbricata effect on tert-BHP induced cytotoxicity
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8.6.15 Hepatoprotective Activity

8.6.15.1 Acute Toxicity

Acetone extract at 2000 mg/kg of root in Swiss albino mice did not indicate any mortality.

8.6.15.2 Effect on Hematological Parameters

Paracetamol causes hepatic injury with a decrease in erythrocytes, leucocytes, and platelets. Administration of acetone extract at 400 mg/kg exhibited increased levels of hematological parameters (Table 8.2).

Table 8.2 Effect on hematological parameters
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8.6.15.3 Effect on Serum Biochemical Markers

Administration of R. imbricata acetone extract at 400 mg/kg decreased the liver markers SGPT (78.92 ± 0.4 U/l), SGOT (89.51 ± 0.1 U/l), and ALP (192.47 ± 0.4 U/l) which was comparable to standard silymarin (25 mg/kg) (Table 8.3).

Table 8.3 Effect on serum biochemical markers
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8.6.15.4 Effect on In Vivo Antioxidant Activity

The use of 400 mg/kg, p.o. acetone extract increases the total proteins, enzymatic antioxidants SOD, and GPx (Table 8.4).

Table 8.4 Effect on in vivo antioxidant activity
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8.6.15.5 Histopathological Examination

Significant reduction of serum enzyme markers, which was elevated by paracetamol, was observed when acetone extract of this plant species was administered at concentrations of 200 and 400 mg/kg, p.o. continuously for 2 weeks and found comparable to silymarin effect. At a dose of 400 mg/kg the acetone extract observed to have the highest protection (Senthilkumar et al. 2014).

8.7 Future Direction

The usage of herbal medicine for the cure of various illnesses has increased greater concern about the safety and least or no side effects. R. imbricata is used in several folklore local medicines. Many scientific studies have established that R. imbricata possesses unique cardioprotective and adaptogenic functions. The importance of this species mentioned in this communication recommended that the species of this plant increase resistance to stress and salidroside, responsible for preventing oxidative stress. Considering the importance of the plant, further biological studies should be conducted to identify the compounds responsible for pharmacological properties, so that the active molecules present in this species of plants could be utilized for future drug development programs.