Anti-nociceptive, anti-inflammatory and sedative activities of the extracts and chemical constituents of Diospyros lotus L.
Diospyros lotus L is traditionally used in various diseases including pain and sleep disorders. The pain and inflammation are the common problems, which are treated with various synthetic analgesic drugs, and associated the side effects. The natural products have gained significant importance over synthetic drugs. The importance of phyto-medicine the current study has been designed with the aim to investigate the analgesic and anti-inflammatory effects of Diospyros lotus and bioassay guided isolation from its crude fractions. Seven known compounds; lupeol (1), 7-methyljuglone (2), [beta]-Sitosterol (3), stigmasterol (4) betulinic acid (5), diospyrin (6; DS) and 8-hydroxyisodiospyrin (7; HDS) which were hitherto unreported from D. lotus. The chloroform fraction (CFDL) and isolated compounds DS and HDS were evaluated for anti-nociceptive, sedative and anti-inflammatory effects. The acetic acid induced writing was significantly (p<0.001) protected by CFDL (72.43%), DS (40.87%) and HDS (65.76%) at higher doses which exhibited peripheral and central analgesic effects in acetic acid and hot-plat pain paradigms. Regarding the anti- inflammatory effect the CFDL (77.43%), DS (80.54%) and HDS (75.87%) protected the carrageenan paw edema after 3rd h. The central analgesic effect was significantly antagonized with naloxone (0.5 mg/kg), showing opiodergic mechanism of action. The CFDL, DS and HDS were also proved sedative in open field animal models. In acute toxicity study the chloroform fraction [CFDL (50,100 and 150 mg/kg)], DS (5 and 10 mg/kg) and HDS (5 and 10 mg/kg) were found safe.
Our study concluded that CFDL, DS and HDS have marked anti-nociceptive, anti-inflammatory and sedative effect. The anti-nociceptive and anti-inflammatory effects of the roots of D. lotus are partially attributed due to the presence of analgesic constituents like diospyrin (DS), 8-hydroxyisodiospyrin (HDS) and strongly supports the ethno-pharmacological uses of D. lotus as anti-nociceptive, anti-inflammatory and sedative.
Pain and inflammation are generally associated with various diseases and usually managed with the use of non-steroidal anti-inflammatory drugs (NSAIDs). In case of chronic inflammatory disorders, the prolonged use of NSAIDs leads to serious side effects including gastric and renal adverse effects (Muhammad et al., 2013a). Commonly the natural medicines are considered to be the safe with minimal side effects (Saeed et al., 2010). Various medicinal plants have been shown to have analgesic and anti-inflammatory effects (Ibrar et al., 2012; Muhammad et al., 2013c), among which Diospyros lotus draw our attention for the exploration of the analgesic and anti-inflammatory activity of its fractions and isolation of active chemical constituents responsible for its activity.
Diospyros lotus belongs to family Ebenaceae which is a deciduous tree, habituated in China and Asia. D. Lotus has been cultivated for its edible fruits which are considered for its medicinal importance. The fruits of D. lotus are used as a sedative, astringent, nutritive, antiseptic, anti-diabetic, antitumor, astringent, laxative, and nutritive as a febrifuge (Uddin et al., 2011a,b). Its fruits are also used for the treatment of diarrhea, dry cough and hypertension (Rashed et al, 2012). Chemical investigation of the fruits led to the identification of some fatty acids, sugars phenolic compounds, and non-volatile acids (Loizzo et al., 2009; Rashed et al., 2012).
The investigation of new, safe, and effective painkiller of natural origin is a big challenge for the researcher to replace the classically used analgesics. Therefore, in the present study, the fractions and bio-assayed guided isolated chemical constituents of D. lotus have been screened for their anti-nociceptive, anti-inflammatory and sedative effects.
Material and methods
Diclofenac sodium (Suzhou Ausun Chemical Co, Lit., China) was used as standard drug. Acetic acid used for induction of pain in writhing test was purchased from (Merck Germany), naloxone (Acent Scientific Company), [tramadol.sup.R] (Searle Pakistan Ltd.). Sterile normal saline was used in all experiments as control while CH[Cl.sub.3] extract was prepared in normal saline.
The diclofenac was used as a reference peripheral analgesic drug which was administered intra-peritonially (i.p.) in dose of 10mg/kg. The CFDL (50, 100 and 150mg/kg), DS (5, 10mg/kg) and HDS (5, 10mg/kg) were dissolved in saline and administered intra-peritonially. Tramadol is used as reference central analgesic drug which was administered intra-peritonially (i.p.) in dose of 30 mg/kg. The standard opioid antagonist naloxone was injected in dose of 0.5 mg/kg subcutaneously. The diazepam reference depressant drug was injected in dose of 0.5 mg/kg i.p. to each animal in positive control group.
BALB/c mice of either sex were used in all experiments. Animals were purchased from the Pharmacology Section of the Department of Pharmacy, University of Peshawar, Peshawar, Pakistan. The animals were maintained in standard laboratory conditions (25[degrees]C and light/dark cycles i.e. 12/12 h) and were fed with standard food and water ad libitum. The experimental protocols were approved by the ethical committee of the Pharmacy Department, University of Peshawar, Peshawar, Pakistan.
Roots of Diospyros lotus were collected from Razagram, Dir, KPK, Pakistan, in May 2009. The sample was authenticated by Dr. Abdur Rashid, Taxonomist; Department Botany, University of Peshawar, Pakistan, where a voucher specimen (Bot. 20036(PUP) has been deposited at the Herbarium.
Extraction and isolation
Shade-dried roots of D. lotus (14 kg) were powdered and then kept at room temperature in MeOH for six days with continuous stirring by simple percolation. The combined extract was concentrated by evaporating solvents using rotary vacuum evaporator under reduced pressure at temperature below 55[degrees]C. A dark red residue (202 g) was obtained which was suspended in water and successively partitioned with n-hexane, CH[Cl.sub.3], EtOAc and n-BuOH as per standard protocol (Uddin et al., 2012a,b). The chloroform fraction (30 g) was subjected to column chromatography on silica gel (Merck Silica gel 60 (0.063-0.200 mm), 5 [right arrow] 60 cm) and eluted with n-hexane-ethyl acetate (100:0 [right arrow] 0:100) as solvent system. As a results, various fractions were obtained and combined on the bases of TLC profile to ultimate afford 105 sub fractions (RF1-RF105). Elution of the chromatogram with n-hexane-ethyl acetate (100:0 [right arrow] 10:100) gave a reddish color oil of fatty acid residues which is followed by 1.25 g of colorless needles that were identified as lupeol (1) (m.p. 210-212[degrees]C) (Lee et al., 2001; Gu et al., 2004) and a white crystal 7-methyljuglone (2) (m.p. 113-116[degrees]C) (Higa et al., 1989), while at polarity range (100:0 [right arrow] 12:100), followed by [beta]-sitosterol (3) (m.p. 137-140[degrees]C) crystal-needles, stigmasterol (4), (m.p. 162-165[degrees]C) (Saluja, 2011; Uddin et al., 2011a) white powder. Betulinic acid (m.p. 317-319[degrees]C) (5) (Ahmad and Atta-ur-Rahman 1994) was obtained as a white crystal at (100:0 [right arrow] 16:100) (Fig. 1). On eluting the column with n-hexane-ethyl acetate (100:0 [right arrow] 20:80) result the isolation of a violet red fraction and revealed two compounds on TLC, which was purified by preparative chromatography and furnish the isolation of a 8-Hydroxyisodiospyrin (6) (m.p. 275-276[degrees]C, 1.4g) (Baker et al., 1998) (Fig. 1). Fraction RF-20 obtained at (100: [right arrow] 15:100) contained red crystals of various sizes, shape and was separated from the solution by washing with n-hexane. To obtain pure and larger crystals, these crystals were re-grown from a mixture of n-hexane-chloroform and thus obtained orange-red crystalline diospyrin (7) (m.p. 252-255[degrees]C, 1.4g) (Fig. 1) (Ferreira et al., 1974).
Acute toxicity test
The acute toxicity test for CFDL, DS and HDS was carried out to evaluate any possible toxicity. BALB/c mice (n = 6) of either sex were tested by administering different doses (500, 1000 and 2000 mg/kg) of CFDL, DS and HDS (25 and 50 mg/kg), while the control group received distilled water (1 ml/kg). All the groups were observed for any gross effect for first 4 h and then mortality was observed after 24 h (Muhammad et al., 2013b).
Acetic acid induced writhing test
BALB/c mice of either sex (n = 6) weighing 18-22 g were used. All animals were withdrawn from food 2 h before the start of experiment and were divided in various groups. Group I was injected with normal saline (10 ml/kg) as control, Group II received standard drug diclofenac sodium (10 mg/kg) while the remaining groups were injected with CFDL (50, 100 and 150 mg/kg i.p.), DS and HDS (5 and 10 mg/kg, i.p.) respectively. After 30 min of the above treatment animals were treated i.p. with 1% acetic acid. The number of abdominal constrictions (writhes) was counted after 5 min of acetic acid injection for the period of 10 min (Muhammad et al., 2012, 2013a).
Hot plat test
BALB/c mice of either sex (n = 6) weighing 18-22g were acclimatized to laboratory conditions one hour before the start of experiment with food and water available ad libitum. Animals were then subjected to pre-testing on hot plate (Havard apparatus) maintained at 55 [+ or -]0.1[degrees]C. Animals having latency time greater than 15 s on hot plate during pre-testing were rejected (latency time). All the animals were divided in various groups each of six mice. Group 1 was treated with saline (10 ml/kg), group II was treated with [Tramadol.sup.R] (30 mg/kg i.p.) while the remaining groups were injected with CFDL (50, 100 and 150 mg/kg i.p.), DS and HDO (5 and 10 mg/kg, i.p.), respectively. After 30 min of treatment the animals were placed on hot plate and the latency time (time for which mouse remains on the hot plate (55[+ or -]0.1[degrees]C) without licking or flicking of hind limb or jumping) was measured in seconds. In order to prevent the tissue damage a cut-off time of 30 s were imposed for all animals. To find out the opiodergic mechanism in the analgesic activity of CFDL, DS and HDS some groups were treated with naloxone (0.5 mg/kg s.c.) and after 10 min these groups were treated with CFDL (100 and 150 mg/kg i.p.), DS, HDS (10 mg/kg, i.p.) and [Tramadol.sup.R] (30 mg/kg i.p.), after 10 min of naloxone injection. The latency time for all groups was recorded at 0, 30, 60, 90 and 120 min. Percent analgesia was calculated using the following formula (Muhammad et al., 2012).
% Analgesia = Test latency - control latency/Cut-off time--control latency x 100
The anti-inflammatory activity was performed on rat of either sex (25-30 g). The animals were randomly divided in various groups each of six animals (Khan et al., 2009). Group I was treated with normal saline (10ml/kg), group 11 with diclofenac sodium (10 mg/kg), rest of the groups were treated with CFDL, DS and HDS at the same dose as in case of analgesic activity. After 30 min of the above intra-peritoneal administration, carrageenan (1%, 0.05 ml) was injected subcutaneously in the sub plantar tissue of the right hind paw of each mouse. The inflammation was measured using Plethysmometer (LE 7500 plan lab S.L) immediately after injection of carrageenan and then after 1, 2, 3,4 and 5 h of carrageenan injection. The average foot swelling in drug treated animals as well as standard was compared with that of control and the percent inhibition (anti-inflammatory activity) of edema was determined using the formula.
Percent inhibition = A - B/A x 100,
where A represent edema volume of control and B as paw edema of tested group.
Open field test
The apparatus used for this activity was consisted of an area of white wood (150-cm diameter) enclosed by stainless steel walls and divided in 19 squares by black lines. The open field was placed inside a light and sound-attenuated room. BALB/C mice of either sex (n = 6) weighing 22 [+ or -] 2 g were used in this study. Animals were acclimatized under red light (40 Watt red bulb) one hour before the start of experiment in laboratory with food and water available ad libitum. The animals were treated with saline, diazepam (reference drug, 0.5 mg/kg i.p.), CFDL (50,100 and 150 mg/kg i.p.), DS and HDS (5 and 10 mg/kg, i.p.). After 30 min of the treatment each animal was placed in the center of the box and the numbers of lines crossed were counted for each mouse (Muhammad et al., 2013b).
Results are expressed as mean [+ or -] S.E.M. One-way ANOVA was used for comparison test of significant differences among groups followed by Dunnet's multiple comparison post-test. A level of significance (p < 0.05 or 0.01) was considered for each test.
The acute toxicity of CFDL fraction of D. lotus was assessed in dose of 500,1000 and 2000 mg/kg. During 24 h assessment of the acute behavioral toxicity, no mortality was observed, however the animals exhibited slight sedation after one hour of the CFDL injection. The treatment dose range of 25-50 mg/kg of DS or HDS exhibited slight sedation with no mortality during 24 h of assessment.
Analgesic effect of D. lotus
Peripheral analgesic effect in acetic acid induced writhing test
The acetic acid induced writing was markedly protected by CFDL as presented in Fig. 2. The CFDL significantly inhibited the writhing dose dependently with the percent inhibition value 33.54% (p<0.05), 50.87% (p<0.05), 72.83% (p< 0.001) at the dose of 50, 100, 150 mg/kg respectively. In case of DS and HDS treatments significant effect (p<0.05) was observed at the tested dose of 5 mg/kg [25.98% and 30.85% for DS and HDS respectively] and 10 mg/kg (40.87 and 65.76% for DS and HDS respectively), however none of the isolated compound exhibited better analgesic effect than that of CFDL.
Central analgesic effect in hot plat test
The crude extract and isolated compounds (DS and HDS) demonstrated significant and dose dependent analgesic effect in thermal induced pain model (hot plate) demonstrated in Table 1. The CFDL significantly increased (p < 0.05) the latency time in tested mice showing the significant analgesic effect in all corresponding doses. The maximum analgesic effect was exhibited after 60 min of post treatment in all treatment groups. The analgesic effect of both DS and HDS significantly increased (p < 0.05) the latency time in a dose dependent manner. The effect of HDS was more than DS and lesser than CFDL. The analgesic effect was significantly antagonized by the injection of naloxone and the latency time was reversed by naloxone in case of CFDL, DS, HDS and [tramadol.sup.R].
Anti-inflammatory effect of D. lotus
The anti-inflammatory effect of CFDL, DS and HDS were dose dependent and diclofenac was used as reference drug as demonstrated in Figs. 3-5. The maximum percent inhibition (58-78.43%) of paw edema was observed after 3rd h of carrageenan injection in CFDL treated animals (Fig. 3). In comparison to the saline treated group, the CFDL exhibited significant inhibition (p < 0.05-0.01) of paw edema right after the first hour of carrageenan injection and onward. Furthermore the paw edema inhibition was observed to be dose dependent.
The DS treatment exhibited maximum paw edema inhibition at third hour of carrageenan injection (40%, 78% for 5 and 10 mg/kg of DS respectively) (Fig. 4). The inhibition was dose dependent and significant (p < 0.05) after one hour and onward in case of 10 mg/kg DS treatment, however 5 mg/kg DS treatment exhibited significant inhibition (p<0.05) after third hour of carrageenan injection.
In the HDS treated animals exhibited the paw edema inhibition and after three hours of carrageenan injection the inhibition was (60% for 5 mg/kg and 78% for 10 mg/kg) (Fig. 5). It was almost similar or a little greater activity than DS. The paw edema inhibition was significant after 1 h of carrageenan injection in the animals treated with 10 mg/kg HDS and significance was increased onward to 1 h. The effect was observed to be dose dependent.
The results of open field i.e., sedative effect are presented in Table 2. The reference drug i.e., diazepam exhibited maximum sedation which was significantly (p < 0.001) different from saline control group. The CFDL was proved as mild sedative (p < 0.05) in treatment dose of 100 and 150 mg/kg compared to the isolated compounds (DS and HDS). The isolated compound DS and HDS exhibited significant (p < 0.01) sedative effect in dose of 5 and 10 mg/kg however these should be consider mild to moderate sedative as the sedation induced was for less than diazepam.
The pain is mostly managed with the classical analgesics including non-steroidal anti-inflammatory drugs (NSAIDs) however in chronic pain conditions (NSAIDs) are used for prolong time which ultimately leads to severe toxicities including GIT ulceration and kidney dysfunction. Thus for management of chronic pain investigation of natural remedies is required to efficiently control the pain with least side effects. In the present modern scientific era, the use of herbal remedies has surpassed the allopathic medicine in the respect of safety and efficiency. Therefore after a thorough literature survey, we selected D. lotus plant to be investigated as a remedy for pain and inflammation. Furthermore, the active chloroform fraction of the plant is subjected to the bioassay guided isolation for identification of its active constituents. The chloroform fraction of D. lotus roots leaded to the isolation of lupeol (1), 7methyljuglone (2), [beta]-sitosterol (3), stigmasterol (4), betulinic acid (5), diospyrin) (6; DS) and 8-hydroxyisodiospyrin (7; HDS) which were hitherto unreported from this species. The first five compounds are previously reported from other plants and evaluated for their activity however the said activities of DS and HDS were not previously reported.
Acetic acid-induced writhing is a well recommended model in evaluating medicinal agents for their analgesic property. The pain induction caused by liberating endogenous substances as well as some other pain mediators such as arachidonic acid via cyclooxygenase, and prostaglandin biosynthesis (Duarte et al., 1988; Khan et al., 2010). This pain paradigm is widely used for the assessment of peripheral analgesic activity due to its sensitivity and response to the compounds at a dose which is not effective in other methods. The local peritoneal receptor could be the cause of abdominal writhings (Mbiantcha et al., 2011). Pain sensation in acetic acid induced writhing paradigm is elicited by producing localized inflammatory response due to release of free arachidonic acid from tissue phospholipids via cyclooxygenase (COX), and producing prostaglandin specifically PGE2 and PGF2[alpha], the level of lipoxygenase products may also increases in peritoneal fluids (Duarte et al., 1988; Khan et al., 2010). These prostaglandin and lipoxygenase products cause inflammation and pain by increasing capillary permeability. The substance inhibiting the writhing will have analgesic effect preferably by inhibition of prostaglandin synthesis, a peripheral mechanism of pain inhibition (Duarte et al., 1988). In our study, the CFDL fraction, DS and HDS exhibited a prominent inhibition of writhing reflux in acetic acid-induced abdominal constriction assay. These findings strongly recommend that the plant and its isolated compounds (DS and HDS) have peripheral analgesic activity. Thermal nociception model such as hot plat test was used to evaluate central analgesic activity of D. lotus roots. CFDL showed significant (P<0.01) analgesic effect in both the hot plat test and acetic acid induced writhing test, implicating both spinal and supraspinal analgesic pathways. In these pain paradigms tramadolR, which is similar to the action of opioid agonists (e.g. morphine), raised the pain threshold level within 30 min of administration. In contrast, CFDL, DS and HDS showed maximum analgesic effect after 60 min of administration. This difference in the maximum analgesic point could be explained by difference in the metabolic rate of each drug or may be the potency of each drug as the analgesic potential of [tramadol.sup.R] is higher than our tested samples. When the non-selective opioid receptor antagonist naloxone was applied, the analgesic effect of CFDL, DS and HDS was also antagonized by naloxone after 30 min of administration which led to the conclusion that the analgesic effect of this extract is due to activation of the opioid receptor stimulation. Furthermore the extract and isolated compounds reduced the locomotive activities in comparison with control group. The reduction in the frequency and amplitude of motion could be attributed to the sedative effect of the extract (Thakur and Mengi, 2005). The isolation of analgesic compounds like lupeol, [beta]-sitosterol, stigmasterol, betulinic acid (Geetha and Varalakshmi, 2001; Gupta et al., 2008) strongly supports the analgesic profile of this valuable plant.
Our study concluded and confirmed that the roots of D. lotus are efficient herbal medicine for subsiding the pain and inflammation. The chloroform fraction of D. lotus (CFDL) and its isolated constituents Diospyrin (DS) (6) and 8-hydroxyisodiospyrin (HDS) (7) exhibited marked anti-nociceptive, anti-inflammatory and sedative effects. The central analgesic effect of D. lotus might be produced through opiodergic mechanism and can be antagonized by naloxone. The anti-nociceptive and anti-inflammatory effects of D. lotus roots are partially attributed due to the presence of analgesic constituents like DS, HDS. This study strongly supports and validates the ethno-pharmacological uses of D. lotus as antinociceptive, anti-inflammatory and sedative activities.
Received 24 January 2014
Accepted 2 March 2014
The authors declared that they have no competing interests. Acknowledgment
The authors are grateful for the financial supported by Higher Education Commission of Pakistan under the Grant number 112-26510-2PS1-258.
Ahmad, V.U., Atta-ur-Rahman, 1994. Handbook of Natural Products Data: Penta-cyclic Triterpenoids. Elsevier, Amsterdam, pp. 1102-1104.
Baker, R.W., Liu, S., Sargent, M.V., 1998. Synthesis and absolute configuration of axially chiral binaphthoquinones. Australian Journal of Chemistry 51, 255-266.
Duarte, I., Nakamura, M., Ferreira, S., 1988. Participation of the sympathetic system in acetic acid-induced writhing in mice. Brazilian Journal of Medical and Biological Research 21, 341.
Ferreira, M.A., Aurea Cruz Costa, M., Alves, A.C., Lopes, M.H., 1974. Naphthoquinones from Euclea pseudebenus. Phytochemistry 13, 1587-1589.
Geetha, T., Varalakshmi, P., 2001. Anti-inflammatory activity of lupeol and lupeol linoleate in rats. Journal of Ethnopharmacology 76, 77-80.
Gu, J.Q., Graf, T.N., Lee, D., Chai, H.B., Mi, Q., Kardono, L.B., Setyowati, F.M., Ismail, R., Riswan, S., Farnsworth, N.R., Cordell, G.A., Pezzuto, J.M., Swanson, S.M., Kroll, D.J., Falkinham 3rd, J.O., Wall, M.E., Wani, M.C., Kinghorn, A.D., Oberlies, N.H., 2004. Cytotoxic and antimicrobial constituents of the bark of Diospyros maritima collected in two geographical locations in Indonesia. Journal of Natural Products 67, 1156-1161.
Gupta, M., Nath, R., Srivastava, N., Shanker, K., Kishor, K., Bhargava, K., 2008. Anti-inflammatory and antipyretic activities of [beta]-sitosterol. Planta Medica 39, 157-163.
Higa, M., Ogihara, K., Yogi, S., 1989. Bioactive naphthoquinone derivatives from Diospyros maritima Blume. Chemical and Pharmaceutical Bulletin 46, 1189-1193.
Ibrar, M., Muhammad, N., Barkatullah, H.K., Jahan, F., Ashraf, N., 2012. Antinociceptive and anticonvulsant activities of essential oils of Zanthoxylum armatum. Phytopharmacology 3 (191), 198.
Khan, I., Nisar, M., Ebad, F., Nadeem, S., Saeed, M., Khan, H., 2009. Anti-inflammatory activities of sieboldogenin from Smilax china Linn.: experimental and computational studies. Journal of Ethnopharmacology 121, 175-177.
Khan, H., Saeed, M., Gilani, A.U.H., Khan, M.A., Dar, A., Khan, I., 2010. The antinociceptive activity of Polygonatum verticillatum rhizomes in pain models. Journal of Ethnopharmacology 127, 521-527.
Lee, S.S., Chang, S.M., Chen, C.H., 2001. Chemical constituents from Alseodaphne andersonii. Journal of Natural Products 64, 1548-1551.
Loizzo, M.R., Said, A., Tundis, R., Hawas, U.W., Rashed, K., Menichini, F., Frega, N.G., Menichini, F., 2009. Antioxidant and antiproliferative activity of Diospyros lotus L. extract and isolated compounds. Plant Foods for Human Nutrition 64, 264-270.
Mbiantcha, M., Kamanyi, A., Teponno, R., Tapondjou, A., Watcho, P., Nguelefack, T., 2011. Analgesic and anti-inflammatory properties of extracts from the bulbils of Dioscorea bulbifera L. var sativa (Dioscoreaceae) in mice and rats. Evidence-based Complementary and Alternative Medicine: eCAM 2011.
Muhammad, N., Saeed, M., Khan, H., 2012. Antipyretic, analgesic and anti-inflammatory activity of Viola betonicifolia whole plant. BMC Complementary and Alternative Medicine 12,59.
Muhammad, N., Saeed, M., Gilani, S.N., 2013a. Analgesic and anti-inflammatory profile of n-hexane fraction of Viola betonicifolia. Tropical Journal of Pharmaceutical Research 11, 963-969.
Muhammad, N., Saeed, M., Khan, H., Haq, I., 2013b. Evaluation of n-hexane extract of Viola betonicifolia for its neuropharmacological properties. Journal of Natural Medicines 67, 1-8.
Muhammad, N., Saeed, M., Khan, H., Raziq, N., Halimi, S.M.A., 2013c. Antipyretic and anticonvulsant activity of n-hexane fraction of Viola betonicifolia. Asian Pacific Journal of Tropical Biomedicine 3, 280-283.
Rashed, K., Luo, Z.X., Zheng, M.Y., 2012. Anti-HIV-1 activity of phenolic compounds isolated from Diospyros lotus fruits. Phytopharmacology 3, 199-207.
Saeed, M., Muhammad, N., Khan, H., Khan, S., 2010. Analysis of toxic heavy metals in branded Pakistani herbal products. Journal of the Chemical Society of Pakistan 32, 471.
Saluja, K.A.A., 2011. Isolation of stigmasterol and 3-sitosterol from petrolum ether extract of parts of ageratum of Ageratum conyzoides. International Journal of Pharmacy and Pharmaceutical Sciences 3, 9496.
Thakur, V., Mengi, S., 2005. Neuropharmacological profile of Eclipta alba (Linn.) Hassk. Journal of Ethnopharmacology 102, 23-31.
Uddin, G., Waliullah, Rauf, A., Siddiqui, B.S., Ahmed, A., Bibi, C., Qaisar, M., Azam, S., 2011a. Phytochemical screening and antimicrobial activity of cornus macrophylla wall, ex Roxb. Middle-East Journal of Scientific Research 9, 516-519.
Uddin, G., Rauf, A., Siddiqui, B.S., Shah, S.Q., 2011b. Preliminary comparative phyto-chemical screening of Diospyros lotus Stewart. Middle-East Journal of Scientific Research 10,78-81.
Uddin, G., Rauf, A., Akhtar, S., 2012a. Studies on chemical constituents, phytochemical profile and pharmacological action Datura alba. Middle-East Journal of Medicanal Plants Research 1, 14-18.
Uddin, G., Rauf, A., Arfan, M., Waliullah, Khan, I., Ali, M., Taimur, M., ur-Rehman, I., Samiullah, 2012b. Pistagremic acid a new leishmanicidal triterpene isolated from Pistacia integerrima Stewart. Journal of Enzyme Inhibition and Medicinal Chemistry 27, 646-648.
Ghias Uddin (a), *, Abdur Rauf (a), Bina S. Siddiqui (c), Naveed Muhammad (b), Ajmal Khan (c), Syed Uzair Ali Shah (c)
(a) Institute of Chemical Sciences, University of Peshawar, Peshawar 25120, KPK, Pakistan
(b) Department of Pharmacy, Abdul Wali Khan University, Mardan, Pakistan
(c) H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan
* Corresponding author. Cell No: +923453256686, Tel.: +92 919216652; fax: +92 91 9216652.
E-mail addresses: firstname.lastname@example.org, email@example.com (G. Uddin).
Table 1 Analgesic effect of CFDL, DS and HDS assessed by using hot plate model. Treatment Group (kg) 30 min 60 min Saline 10 ml 9.20 [+ or -] 0.08 9.18 [+ or -] 0.09 Tramadol 30 mg 24.67 [+ or -] 0.07 ** 26.08 [+ or -] 0.100 *** CFDL 50 mg 11.92 [+ or -] 2.89 * 12.22 [+ or -] 2.09 * 100 mg 13.33 [+ or -] 2.33 * 14.88 [+ or -] 2.87 ** DS 150 mg 19.70 [+ or -] 2.09 * 24.75 [+ or -] 2.91 ** 5 mg 11.92 [+ or -] 0.44 * 12.88 [+ or -] 0.90 * HDS 10 mg 12.92 [+ or -] 0.44 15.20 [+ or -] 1.11 * 5 mg 12.00 [+ or -] 01.00 * 12.90 [+ or -] 1.23 * 10 mg 13.00 [+ or -] 0.95 * 14.89 [+ or -] 1.11 * Analgesic effect antagonized by naloxone (0.5 mg/kg S.C.) CFDL 100 mg 10.90 [+ or -] 2.56 ** 10.80 [+ or -] 0.39 ** DS 150 mg 10.99 [+ or -] 2.00 ** 10.80 [+ or -] 2.91 ** 10 mg 11.00 [+ or -] 01.00 * 11.90 [+ or -] 1.23 * HDS 10 mg 12.00 [+ or -] 0.78 * 13.89 [+ or -] 0.98 * Tramadol 30 mg 10.22 ** 10.02 *** [+ or -] 0.05 [+ or -] 0.09 Treatment Group (kg) 90 min 120 min Saline 10 ml 9.20 [+ or -] 0.03 9.17 [+ or -] 0.11 Tramadol 30 mg 25.77 [+ or -] 0.11 *** 25.66 [+ or -] 0.45 *** CFDL 50 mg 12.89 [+ or -] 2.55 * 12.66 [+ or -] 2.98 * 100 mg 14.20 [+ or -] 2.90 ** 14.09 [+ or -] 1.99 ** DS 150 mg 23.98 [+ or -] 1.76 ** 23.60 [+ or -] 2.87 ** 5 mg 12.78 [+ or -] 0.87* 12.58 [+ or -] 1.00 * HDS 10 mg 15.00 [+ or -] 0.77 ** 14.80 [+ or -] 0.99 ** 5 mg 12.80 [+ or -] 1.90 * 12.70 [+ or -] 1.22 * 10 mg 14.70 [+ or -] 0.89 ** 14.60 [+ or -] 0.87 ** Analgesic effect antagonized by naloxone (0.5 mg/kg S.C.) CFDL 100 mg 10.95 [+ or -] 2.98 ** 10.98 [+ or -] 1.99 ** DS 150 mg 10.85 [+ or -] 1.88 ** 10.87 [+ or -] 1.49 ** 10 mg 11.80 [+ or -] 1.90 * 11.70 [+ or -] 1.22 * HDS 10 mg 13.70 [+ or -] 0.39 ** 13.60 [+ or -] 0.57 * Tramadol 30 mg 10.24 *** 10.05 *** [+ or -] 0.03 [+ or -] 0.00 Values are reported as mean [+ or -] S.E.M. for group of six animals. The data was analyzed by ANOVA followed by Dunnett's test * p < 0.05 statistically significant value from control. ** p<0.01 statistically significant value from control. *** p< 0.001 statistically significant value from control. Table 2 Sedative effect of CFDL, DS and HDS. Treatment Dose(mg/kg) No of line crossed in 10 min Saline lOml/kg 130.54 [+ or -] 0.98 Diazepam 0.5 6.87 [+ or -] 0.11 *** CFDL 50 125.34 [+ or -]2.98 100 120.90 [+ or -] 2.00 * 150 112.80 [+ or -] 2.90 * DS 5 100.34 [+ or -]0.87 ** 10 91.98 [+ or -] 1.09 *** HDS 5 99.89 [+ or -] 1.33 ** 10 90.02 [+ or -] 2.98 *** The values represent the number of lines crossed by animal in box, 30 min after treatment with saline (10 ml/kg, control), diazepam. CFDL, DS or HDS. Data presented as mean [+ or -] S.E.M. for n = 6. (a) p < 0.05 level of significant compared with control. (b) p < 0.01 level of significant compared with control. (c) p < 0.001 level of significant compared with control.