Beneficial Effects of Trillium govanianum Rhizomes in Pain and Inflammation

Trillium govanianum rhizome is used as an analgesic and anti-inflammatory remedy in traditional medicine in northern Pakistan. In an attempt to establish its medicinal value, the present research evaluated the analgesic and anti-inflammatory potential of T. govanianum. The in vivo anti-inflammatory activity of extract and fractions was investigated in the carrageenan induced paw edema assay. The in vitro suppression of oxidative burst of extract, fractions and isolated compounds was assessed through luminol-enhanced chemiluminescence assay. The in vivo analgesic activity was assayed in chemical and thermal induced nociceptive pain models. The crude methanol extract and its solvent fractions showed anti-inflammatory and analgesic responses, exhibited by significant amelioration of paw edema and relieve of the tonic visceral chemical and acute phasic thermal nociception. In the oxidative burst assay, based on IC50, the crude methanol extract and n-butanol soluble fraction produced a significant inhibition, followed by chloroform and hexane soluble fractions as compared to ibuprofen. Similarly, the isolated compounds pennogenin and borassoside E exhibited significant level of oxidative burst suppressive activity. The in vivo anti-inflammatory and analgesic activities as well as the in vitro inhibition of oxidative burst validated the traditional use of T. govanianum rhizomes as a phytotherapeutic remedy for both inflammatory conditions and pain. The observed activities might be attributed to the presence of steroids and steroid-based compounds. Therefore, the rhizomes of this plant species could serve as potential novel source of compounds effective for alleviating pain and inflammation.


Introduction
It is highly desired to discover excellent remedies for diseases that are economical, having no or low side effects, potent and efficacious in various pathological conditions.For discovering such products, medicinal plants and herbal medicines can be the best choice as plants produce a wide range of bioactive compounds, making them a rich source of different types of medicines [1].Numerous drugs have entered international pharmacopoeias through ethnopharmacological and traditional medicine studies [2].Based on folkloric use, research on medicinal plants has proved the presence of valuable pharmacologically active compounds with anti-cancer, antiparasitic, antifungal, antibacterial, analgesics, and anti-inflammatory properties [3][4][5].In developing countries, majority of population depends largely on traditional system of medicines because of their low cost and ease of availability [6].
At present, available drugs for the management of pain and inflammatory conditions are either narcotic, non-narcotic analgesics or corticosteroids, possessing well known side and adverse effects [7].Furthermore, synthetic drugs are very expensive to develop since, for the successful introduction of a new drug product hundreds and thousands of compounds are to be synthesized, screened and tested which involves a huge expense as well as time.On the other side many medicines of plant origin have been used since long with minimum or none side and adverse effects [8].It is, therefore, inevitable to search medicinal plants for effective analgesic and anti-inflammatory molecules with significant therapeutic potentials.
Trillium govanianum belongs to the genus Trillium (family: Melanthiaceae alt.Trilliaceae) is an indigenous medicinal plant of Pakistan, found at an altitudinal range of 2500-3800 m [9].In traditional medicines, rhizomes of this plant species are used for treating wounds, dysentery, skin boils, infections, and menstrual and sexual disorders [10][11][12].The genus Trillium consists of 31 species, widely distributed from the western Himalayas to Japan, China, Kamchatka (Russia) and North America [13] and is an important source of bioactive compounds of different classes like steroids, glycosides, terpenoids, sterols, saponins, sapogenins and flavonoids [14][15][16].Literature studies indicate that plant species of this genus have been extensively used as remedy for various diseases like T. tschonoskii has been traditionally used in China, for at least one thousand years, for treatment of neurasthenia, giddiness, headache, removing carbuncles and ameliorating pains [17][18][19].The marked inhibitory activity against COX-2 production in macrophagocytes of the mouse abdominal cavity by isolated compounds from T. tschonoskii has also been reported [20].It has also been described that the ethanol, ethyl acetate and n-butanol extracts of T. tschonoskii significantly suppress carrageenan-induced edema in rats [21].The rhizomes of T. erectum called 'beth root' have been used in folk medicine for the treatment of hemorrhages of uterus, urinary tract and lungs [22].
Phytochemical analyses of T. govanianum rhizomes have resulted in isolation of steroids and saponins from the chloroform and n-butanol soluble fractions [23].Moreover, antifungal and anticancer activities in addition to ethnomedicinal relevance of T. govanianum have also been recently reported [24,25].However, to best of our knowledge studies concerning the therapeutic efficacy of T. govanianum as an analgesic and anti-inflammatory agent have not been carried out.Therefore, in present study, we evaluated the crude methanol extract and solvent soluble fractions thereof for analgesic and anti-inflammatory activities, using in vivo animal models, while the isolated compounds in our previous study [23] were tested for inhibition of oxidative burst using luminol-enhanced chemiluminescence assay.

In Vivo Toxicity Test
No overt behavioral change was reported during an observation time of 2 h post crude methanol extract administration.As shown in Table 1, maximum mortality was observed at a dose of 6000 mg/kg while no lethality was observed at 500 mg/kg dose.From the LD 50 value (2030.4mg/kg), the crude methanolic extract is considered as safe to the maximum of tested dose used in this study.

In Vitro Toxicity Test
The crude methanolic extract and its fractions were tested in vitro against NIH 3T3 mouse embryo fibroblast cell line to ascertain their cytotoxicity.As shown in Table 2, the IC50 values for the in vitro assay were in following order: cycloheximide < BuOH-fr < Aq-fr < Chl-fr < EtOAc fraction < Hex-fr < MeOH-ext.The cytotoxicity decreased in the following rank order: MeOH-ext > Hex-fr > EtOAc-fr > Chl-fr > Aq-fr > BuOH-fr > cycloheximide.The standard cycloheximide was more cytotoxic to fibroblasts compared to crude extract and its fractions.

Anti-Inflammatory Activity
The anti-inflammatory response of T. govanianum rhizomes methanol extract (MeOH-ext) and fractions thereof in carrageenan-induced paw edema model is presented in Table 3 The results indicate that MeOH-ext and its fractions at 50, 100 and 200 mg/kg exhibit significant anti-inflammatory activity comparable to the standard anti-inflammatory drug, diclofenac sodium.
The MeOH-ext and its fractions at doses of 100 and 200 mg/kg showed an anti-inflammatory activity that became significant (p < 0.01) in the second phase of inflammation, i.e., 2 h after the injection of carrageenan.The anti-inflammatory effect remained significant in the second phase with a maximum percent inhibitions of 64.6 ± 4.0, 63.5 ± 0.5, 47.5 ± 0.5 and 72.6 ± 3.9 by MeOH-ext, chloroform, ethyl acetate and n-butanol fractions respectively.The MeOH-ext and its fractions showed a slightly weaker activity in early phase of inflammation (1-2 h), though n-butanol fraction was active at a dose of 100 mg/kg.
In chemiluminescence assay, the in vitro inhibitory effects on the release of reactive oxygen species (ROS) from whole blood by MeOH-ext and its fractions are presented in Table 4.The results show that n-butanol fraction exhibit significant inhibition of oxidative burst for whole blood followed by MeOH-ext with IC 50 ± SD of 16.5 ± 7.5 and 30.8 ± 7.0 µg/mL respectively.11.2 ± 1.9 Values represent mean ± SEM of three independent experiments.
Based on these results, isolated compounds i.e., diosgenin, pennogenin and borassoside E (Figure 1) from T. govanianum rhizomes were screened for inhibitory effects on release of ROS from whole blood (Table 4).Pennogenin exhibited significant in vitro inhibitory effect by controlling release of ROS with IC 50 of 5.0 ± 0.8 µg/mL, while borassoside E and diosgenin showed weak inhibition.
Inflammation and reactive oxygen species (ROS) has a relationship of mutual promotion.ROS are associated with inflammatory response and may contribute tissue damages [26].In addition, oxidative stress is thought to play an important role in the pathogenesis of many chronic inflammatory diseases by different molecular mechanisms [27].Numerous studies have shown that production of reactive species such as ROS, reactive nitrogen species (RNS) and hypochlorous acid (HOCl) occur at the site of inflammation, thus exacerbating tissue damage as well as the inflammatory cascade [28,29].
Drugs that inhibit the formation or release of these toxic reactive species (or detoxify them) are effective in treating a variety of diseases that involves stimulation of immune cells like AIDS, rheumatoid arthritis and cancer [30].Luminol-enhanced chemiluminescence assay is based on the detection of intracellular ROS released by opsonized zymosan activated immune cells.A measurement of chemiluminescence is an efficient and highly sensitive method to investigate different kinds of ROS, and is also a suitable method for detection of superoxide anion in biological systems [31].The carrageenan-induced paw edema model, represent a form of acute inflammation, has been frequently used to assess the anti-inflammatory effects of natural products [32].This is useful model to assess the contribution of mediators involved in vascular changes associated with acute inflammation [33].Within first hour following carrageenan injection, edema is induced by the release of various mediators such as histamine, bradykinin and 5-HT (5-hydroxytriptamine or serotonin), but not through prostaglandins (PG).These mediators, following activation of their receptors on endothelial cells, trigger iNOS (inducible nitric oxide synthase) activation and the generation of nitric oxide (NO), and RNS.In mice, following the intraplantar injection of carrageenan, tissue necrosis factor as well as cytokines such as IL-1 and IL-2 are produced [34].Cyclooxygenase-2 (COX-2) is also induced within 2 h post carrageenan administration [35].The NOS and COX pathways appear to operate together to amplify the inflammatory response.The dual inhibition of NO and PG obtained with NOS inhibitors could account for their marked anti-inflammatory activity [33].Neutrophil infiltration in response to carrageenan and following membrane NADPH oxidase activation generates an oxygen respiratory burst giving rise to oxygen-derived free radicals and eliciting immune cell recruitment, which, in turn, produces tissue damage [36].
The ability of T. govanianum rhizomes MeOH-ext, fractions and isolated compounds thereof to inhibit release of ROS from whole blood after serum opsonized zymosan activation, may be a consequence of either scavenging of ROS during oxidative burst or inhibition of enzymes involved in their production such as NADPH oxidase, superoxide dismutase (SOD), catalases and peroxidases.As there is correlation between in vitro anti oxidative activities and in vivo anti-inflammatory activities [37], thus our in vitro results strongly support significant in vivo anti-inflammatory activity by extract and fractions in the carrageenan-induced paw edema model.Furthermore, diosgenin, a saponin aglycon found in a variety of plants, has anti-inflammatory properties, and it has been reported that diosgenin at dose of 400 µg/kg is responsible for maximum anti-inflammatory effect (82.25%) in carrageenan induced paw edema model [38].Since, diosgenin is one of the major metabolite of T. govanianum rhizomes [23], it confirms our in vitro and in vivo anti-inflammatory results.

Abdominal Constriction Assay (Tonic Visceral Chemically-Induced Nociception)
The results of MeOH-ext and its solvent soluble fractions in tonic visceral chemical-induced nociception assay are presented in Figure 2 (Table S1; Supplementary materials), which indicates that 50 and 100 mg/kg doses significantly attenuate acetic acid induced writhes for Hex-fr (p < 0.01, The carrageenan-induced paw edema model, represent a form of acute inflammation, has been frequently used to assess the anti-inflammatory effects of natural products [32].This is useful model to assess the contribution of mediators involved in vascular changes associated with acute inflammation [33].Within first hour following carrageenan injection, edema is induced by the release of various mediators such as histamine, bradykinin and 5-HT (5-hydroxytriptamine or serotonin), but not through prostaglandins (PG).These mediators, following activation of their receptors on endothelial cells, trigger iNOS (inducible nitric oxide synthase) activation and the generation of nitric oxide (NO), and RNS.In mice, following the intraplantar injection of carrageenan, tissue necrosis factor as well as cytokines such as IL-1 and IL-2 are produced [34].Cyclooxygenase-2 (COX-2) is also induced within 2 h post carrageenan administration [35].The NOS and COX pathways appear to operate together to amplify the inflammatory response.The dual inhibition of NO and PG obtained with NOS inhibitors could account for their marked anti-inflammatory activity [33].Neutrophil infiltration in response to carrageenan and following membrane NADPH oxidase activation generates an oxygen respiratory burst giving rise to oxygen-derived free radicals and eliciting immune cell recruitment, which, in turn, produces tissue damage [36].
The ability of T. govanianum rhizomes MeOH-ext, fractions and isolated compounds thereof to inhibit release of ROS from whole blood after serum opsonized zymosan activation, may be a consequence of either scavenging of ROS during oxidative burst or inhibition of enzymes involved in their production such as NADPH oxidase, superoxide dismutase (SOD), catalases and peroxidases.As there is correlation between in vitro anti oxidative activities and in vivo anti-inflammatory activities [37], thus our in vitro results strongly support significant in vivo anti-inflammatory activity by extract and fractions in the carrageenan-induced paw edema model.Furthermore, diosgenin, a saponin aglycon found in a variety of plants, has anti-inflammatory properties, and it has been reported that diosgenin at dose of 400 µg/kg is responsible for maximum anti-inflammatory effect (82.25%) in carrageenan induced paw edema model [38].Since, diosgenin is one of the major metabolite of T. govanianum rhizomes [23], it confirms our in vitro and in vivo anti-inflammatory results.
Abdominal constriction assay (acetic acid-induced writhing method) is widely used for evaluation of peripheral anti-nociceptive activity [39].In tonic visceral chemical-induced nociception model, injection of acetic acid into the peritoneal cavity of mice has been attributed to the release of arachidonic acid (AA), which contributes in prostaglandin synthesis through cyclooxygenase enzyme [8].The special nerve endings that sense pain are very sensitive to prostaglandin.When prostaglandins are released, nerve endings respond to it through prostaglandin E2 receptor, transmitting the pain and injury messages to brain and induce strong contraction tracked by extension of the hind limbs (writhing).This visceral pain model is simple, reliable and rapid for investigation of peripheral analgesics.p < 0.001), Chl-fr (p < 0.01, p < 0.05), EtOAc-fr (p < 0.01), BuOH-fr (p < 0.001, p < 0.05), Aq-fr (p < 0.001), and MeOH-ext (p < 0.001, p < 0.01), respectively.The antinociceptive activity was comparable to the standard drug diclofenac sodium, which significantly relieved (p < 0.001) the tonic visceral chemical-induced nociceptive pain.
Abdominal constriction assay (acetic acid-induced writhing method) is widely used for evaluation of peripheral anti-nociceptive activity [39].In tonic visceral chemical-induced nociception model, injection of acetic acid into the peritoneal cavity of mice has been attributed to the release of arachidonic acid (AA), which contributes in prostaglandin synthesis through cyclooxygenase enzyme [8].The special nerve endings that sense pain are very sensitive to prostaglandin.When prostaglandins are released, nerve endings respond to it through prostaglandin E2 receptor, transmitting the pain and injury messages to brain and induce strong contraction tracked by extension of the hind limbs (writhing).This visceral pain model is simple, reliable and rapid for investigation of peripheral analgesics.
Hot plate test is one of the most common tests employed in evaluation of central analgesic potential of drugs.The mice paws are very sensitive to heat at temperatures, which are not damaging to skin.Mice show escape responses to the nocifensive stimulus and are manifested as jumping, licking or withdrawal of the paws.These responses take prolonged time to appear after
Hot plate test is one of the most common tests employed in evaluation of central analgesic potential of drugs.The mice paws are very sensitive to heat at temperatures, which are not damaging to skin.Mice show escape responses to the nocifensive stimulus and are manifested as jumping, licking or withdrawal of the paws.These responses take prolonged time to appear after administration of centrally acting analgesic drugs.Thus, the hot plate test model measures different responses to acute nociceptive or non-inflammatory inputs and is one of the models normally used for studying central antinociceptive activity [40].It has been reported that steroidal saponins are among the major chemical constituents in medicinal preparations responsible for most of the anti-inflammatory and analgesic activities.Recent reports also indicate that saponins suppress the expression of iNOS and COX-2, thus resulting in a noticeable lowering of prostaglandin E2 levels [41,42].The phytochemical analysis of T. govanianum rhizomes showed that it is a rich source of steroids and saponins (Figure 1) [36], therefore it is assumed that the observed effects are due to the presence of these metabolites in T. govanianum [43,44].
It is worth mentioning that mice were selected as the species of choice in these specific tests because they are noticeably sensitive not only to opioid-mediated effects, but also to coexistent non-steroidal anti-inflammatory drugs (NSAIDs) activity [45,46].Moreover, the murine abdominal constriction assays as well as the hot plate test detect dose-dependent anti-nociception in this species quite well [47,48].

Extraction and Fractionation
The shade-dried rhizomes of T. govanianum (7 kg) were grounded and extracted with MeOH (40 L) at room temperature, three times for seven days (3 × 40 L).The combined methanolic extract was evaporated to dryness that yielded a brownish gummy residue (512 g).It was further fractionated (solid-liquid partition) into hexane (Hex-fr, 81 g), chloroform (Chl-fr, 94 g), ethyl acetate (EtOAc-fr, It has been reported that steroidal saponins are among the major chemical constituents in medicinal preparations responsible for most of the anti-inflammatory and analgesic activities.Recent reports also indicate that saponins suppress the expression of iNOS and COX-2, thus resulting in a noticeable lowering of prostaglandin E 2 levels [41,42].The phytochemical analysis of T. govanianum rhizomes showed that it is a rich source of steroids and saponins (Figure 1) [36], therefore it is assumed that the observed effects are due to the presence of these metabolites in T. govanianum [43,44].
It is worth mentioning that mice were selected as the species of choice in these specific tests because they are noticeably sensitive not only to opioid-mediated effects, but also to coexistent non-steroidal anti-inflammatory drugs (NSAIDs) activity [45,46].Moreover, the murine abdominal constriction assays as well as the hot plate test detect dose-dependent anti-nociception in this species quite well [47,48].

Extraction and Fractionation
The shade-dried rhizomes of T. govanianum (7 kg) were grounded and extracted with MeOH (40 L) at room temperature, three times for seven days (3 × 40 L).The combined methanolic extract was evaporated to dryness that yielded a brownish gummy residue (512 g).It was further fractionated (solid-liquid partition) into hexane (Hex-fr, 81 g), chloroform (Chl-fr, 94 g), ethyl acetate (EtOAc-fr, 85 g), n-butanol (BuOH-fr, 115 g), and aqueous (Aq-fr, 205 g) fractions.The chloroform and n-butanol fractions were subjected to column chromatography and elution was carried out with mixture of hexane, chloroform, EtOAc and MeOH in increasing order of polarity.The compounds diosgenin and pennogenin were obtained at 20 and 25% EtOAc/hexane solvent system, respectively, while borassoside was obtained at 5% MeOH/EtOAc solvent system.The doses of the extract and its fractions used in the in vivo assays were selected in relation to the usual therapeutic doses of the respective standard drugs (controls) utilized in the pharmacological activities.

Animals
BALB/c mice of either sex (21-35 g) were maintained and acclimatized at 25 ± 2 • C under a 12 h dark/light cycle.Food and water were provided ad libitum.The experimental procedures were approved by the Ethical Committee of the Department of Pharmacy, University of Peshawar, Pakistan (14/EC/Pharm).

In Vivo Toxicity
The acute toxicity test was performed according to the organization for economic co-operation for development (OECD) guidelines for the testing of chemicals, Test No. 423 (OECD guidelines, 2001) and the same methodology is also used for screening of medicinal plants [49].The animals were divided into six groups each consisting of six mice (n = 6).T. govanianum extract was administered in doses of 250, 500, 1000, 1500, 3000 and 6000 mg/kg body weight through an oral gavage tube and the morbidity was observed continuously for the first 2 h and mortality up to 24 h post dose administration [50].The animals were observed for spontaneous activity, aggressiveness, cyanosis, ataxia, tail pinch response, righting reflex, writhing, convulsions, catalepsy and bizarre behavior.The 50% mortality among the animals indicated the toxicity concentration of the extract which was calculated using probit analysis.

In Vitro Toxicity
The in vitro cytotoxic activity of methanol extract and fractions thereof was determined by following MTT assay, with slight modification [51,52], on 3T3 cell lines.

Carrageenan Induced Paw Edema Assay
The anti-inflammatory activity was performed using the carrageenan induced paw edema model in mice [53].Briefly, BALB/c mice (25-30 g) were randomly divided into five groups (n = 6).Group I was treated with normal saline (10 mL/Kg), group II with diclofenac sodium (10 mg/Kg), while the other groups were treated with T. govanianum rhizomes MeOH-ext and its fractions (50, 100 and 200 mg/Kg; p.o.).After 30 min of drug treatment, 0.05 mL low molecular weight carrageenan (MW 20-30 kD, 1% w/v in 0.9% NaCl w/v solution) was injected subcutaneously in the sub plantar tissue of the right hind paw of each mouse.The inflammation was measured using a digital plethysmometer (LE 7500 plan lab S.L) immediately after injection of carrageenan and, then, at 1, 2, 3, 4 and 5 h intervals.The average paw swelling in tested samples as well as standard treated animals was compared with that of control and percent inhibition (anti-inflammatory activity) of edema was determined using the following formula: Percentage of edema inhibition (%) = (VA − VB/VA) × 100 where VA is the edema volume of control and VB is the volume of paw edema in treated group.

Table 1 .
In vivo toxicity test of Trillium govanianum rhizome methanol extract.

Table 2 .
In vitro cytotoxicity assay of Trillium govanianum rhizome methanol extract and its fractions on NIH 3T3 mouse embryo fibroblasts.

Table 3 .
Anti-inflammatory activity of Trillium govanianum rhizome methanol extract and fractions in carrageenan-induced paw edema assay.

Table 4 .
Inhibitory effect on release of ROS by Trillium govanianum methanol extract, fractions and isolated compounds.