INTRODUCTION:

Pain is discomfort commonly associated with tissue damage in the body. Pain is as a result of tissue damage that releases several chemical compounds like prostaglandins, bradykinins, and substance P to the nociceptors, thereby generating this sensation¹. Analgesics are the drugs used to relieve or reduce any pain, and traditional analgesic drugs, especially the opiates and non-steroidal anti-inflammatory drugs, are based on natural products. Nonetheless, there are many synthetic alternatives which have been produced with working in the same manner, closely attached to serious side effects such as ulceration, gastrointestinal bleeding, and risk of addiction and even respiratory distress, somnolence, and nausea². The severity of the pain may vary in the scale between mild, moderate, to severe, and it may also be variable (being sharp, burning, or dull); duration (being short lived, intermittent, or persistent); referral (and whether it is superficial or deep, localised or diffuse). The commonest cause of pain is a direct consequence of an unfavourable incident associated with tissue damage, such as an accident, inflammation, or cancer, but severe pain may have no evident cause (such as trigeminal neuralgia) or persist many years after the original trauma has healed (such as in phantom limb pain). Also, there are some of them that are commonly observed in the patients with pain, and it is hyperalgesia (exaggerated response to a painful stimulus), allodynia (the feeling of pain with a mechanical stimulus that would otherwise be non-painful), and hyperesthesia (pathological responsiveness to sensory stimulation)³. Several issues, like the kind of pain, its cause, and origin, determine the healing of pain. Neurogenic pain may occur as a result of the presence of anxiety, depression, mania, epilepsy, seizures, phobias, among other ailments, and therefore requires the application of neurotherapeutic agents that focus on serotonin/ norepinephrine reuptake inhibitors. Comparatively, other types of pains, such as body aches, arthritic pains, inflammatory pains and trauma pain, are generally treated using common analgesic drugs, such as non-steroidal anti-inflammatory drugs. Opioids have been the most frequently used analgesics in the world, hence the most preferred in the treatment of pain. Their mode of action is the ability to bind to the opioid receptors in the central nervous system (CNS), mimicking the effects of endogenous peptide neurotransmitters called endorphins, enkephalins and dynorphins⁴. A substantial amount of modern-day pharmacological agents have rice out of natural sources. The idea that nature is a source of the new therapeutic agents is not new, since it is several thousands of years old. Many medicines have been discovered as they are found in nature, especially the ones found in traditional medicine. It has been observed that most of the critical pharmaceuticals in use today were derived or isolated by plants which the native people used. It is estimated that the consumption of local medicine and medicinal plants fulfils the need of approximately 60 percent of the world population⁵. A definition of pain has been given by the International Association for the Study of pain (IASP) as: pain is an unpleasant sensory and emotional experience associated with actual or potential tissue damage”. Morphine and aspirin are known recent pain killers. In most cases, both opioids and nonsteroidal anti-inflammatory drugs (NSAIDs) can have a pain alleviation effect in only 50 per cent of people, and only 30 percent of them, at most. Moreover, some of these drugs are highly toxic. Studies show that opiates may cause physical dependence, tolerance, and addiction⁶. Chronic pain is a critical health condition affecting 8 per cent of the population in the world. The victims of such a disease are also worse off physically and mentally, and hence have a diminished quality of life. Reducing moderate to severe acute and chronic pain using existing pharmaceutical treatment is ineffective. The key classes of drugs used in the treatment of pain are opioids, paracetamol, nonsteroidal anti-inflammatory, muscle relaxants, anticonvulsants, and COX2 inhibitors. The side effects that are associated with it are constipation, drowsiness, renal toxicity and exhaustion⁷.This sensation is directly caused by tissue damage since it produces a variety of chemical mediators, such as prostaglandins, bradykinins and substance P which is affected on to the nociceptors to cause this sensation. Nociceptive stimulus travels through small myelinated thin C-fibres up to the central nervous system. It was commonly classified as acute and chronic. Acute pain could be characterized by the fact that, it is the type of pain in which its duration is very brief ranging to an hour or so and the onset is very fast. On the contrary, chronic pain is often associated with persistent pain experienced in a long duration of time ¹. The neurological system forms pain and it is a norm among the human beings in every part of the globe. Pain may be acute (accompanied with damage) or chronic (e.g., caused by rheumatoid arthritis or gout, or in cancer). excessive and prolonged use of over-the-counter (OTC) medication, including aspirin or paracetamol, may lead to gastric ulcers or liver damage⁸. Most pain relief medications have serious physiological side effects that have been observed over the past 30 years. Some of these traditional medicine plants used are analgesics and researchers have checked crude extracts as analgesics ⁹. The world needs to make sure of having alternative sustaining more efficient and with fewer side effects herbal analgesics at an affordable price that can be developed with the help of medicinal plants ¹⁰.

Fig.1 Sansevieria roxburghiana

Fig.2 Sansevieria Roxburghiana Flowers

Sansevieria roxburghiana belongs to the group of Dracaenaceae and it is also referred to as bowstring hemp, piles root, and Jaang Mattai as a Tamil name. Studies conducted on Sansevieria species have proved anti-inflammatory activity, analgesic activity, antioxidant activity and antimicrobial activity. Despite a lot of literature developed across the diseases on the biological activities of the Sansevieria genus, there is not much on the antimicrobial effects of the S. Roxburghiana. The current amount of research was concentrated on the study of antimicrobial qualities observed in the leaves and rhizomes in S. Roxburghiana. Z-get Short-Plants like Sansevieria cylindrica, Sansevieria Ehrenberg, Sansevieria guineensis, Sansevieria longiflora, Sansevieria trifasciata, and Sansevieria zeylanica are used as ornamentals, and they grow very easily with little light and they have long rhizomes and fibrous roots¹¹.

Mechanism Of Analgesic Drugs:

Pain happens when neurotransmitters stimulate the nociceptive receptors. Three receptors which participate in the perception of pain are identified, namely: mu, kappa and delta. These receptors stimulate the synthesis of either prostaglandin I or prostaglandin II (However, at times both). The pain relievers block these receptors selectively or non-specifically at the COX-II receptor. The mechanism of opioids in relieving pain entails their ability to increase the pain threshold within the spinal cord, enabling a person to cope with increased pain threshold ⁴.

Fig.3 Mechanism of Analgesic drugs

MATERIALS AND METHODS:

Plant Material:

For the present investigation, the Sansevieria roxburghiana were collected from the village: Annaram, District: Medak, and were identified at the Botanical Survey of India located at Koti, Hyderabad, Telangana. The identification number 780 for Sansevieria roxburghiana was deposited. The collected plant parts were cleaned and dried for 2 weeks, and ground into a fine powder with the help of an electronic grinder. The powder was stored in an airtight container and kept in a dry place.

Animals: Institutional animal ethical committee (CPCSEA Registration no. 1684/PO/Re/S/13/CPCSEA) and proposal number: IAEC/1684/PEIP/01-01-2025/MICE-24/02. This certifies that the IAEC has approved the animal activity part on 1/03/2025 for the project entitled “Evaluation of phytoconstituents and analgesic activity on Sansevieria roxburghiana leaves by in vivo methods” submitted by Mrs. P. Madhavi for the award of a Bachelor of Pharmacy project under JNTU Hyderabad.

Preparation of Extracts: Leaves of Sansevieria roxburghiana were collected, cleaned, and dried in the shade. The leaves were ground into fine powder. The leaf powder was weighed for the Soxhlet method and extracted with distilled water, with the measurements of 30 grams of plant fine powder with 300 ml of the solvent that are used. In the Soxhlet method, after 12 cycles for 8 hours, the concentrated samples of plant extracts were collected. The aqueous extracts were dried by using a rotary vacuum evaporator¹².

Fig.4 Soxhlation of S. roxburghiana leaves

Fig.5 Soxhlation Apparatus set

Phytochemical Screening:

Phytochemical screening of the extracts was conducted using various qualitative tests that had been identified. The phytochemicals present in Sansevieria roxburghiana are aqueous extracts, common constituents of alkaloids, flavonoids, carbohydrates, saponins, tannins, steroids, and Glycosides are present in aqueous extracts¹².

Physicochemical Tests:

Solubility: The solubility of the Sansevieria roxburghiana aqueous extract is determined by mixing in various volumes and it is soluble in different solvents like ethanol, methanol and water.

Specific Gravity: Specific gravity is a criterion of the quality and purity of the quality and purity of extract. The actual weight of the specific gravity bottle or pycnometer will be considered as W1 and weigh the plant extract, like 2 gm and dissolve it with water and fill with sample pycnometer with and fill the sample and weigh the pycnometer and name it as W3. Weigh the distilled water up to the marking of the pycnometer and weigh and mark as W2, and calculate the result using the formula.

Formula:

Specific Gravity= W3-W1/ W2-W1

S.G = 47.61 -24.15/47.61-24.15= 0.785.

The specific gravity value of the aqueous extract of Sansevieria roxburghiana is 0.785.

Saponification Value: Determine the saponification value, precisely weigh 1 to 2 grams of the plant extract sample into a clean, dry 250 mL conical flask. Add 25 mL of alcoholic 0.5 N potassium hydroxide (KOH) solution. Prepare alcoholic KOH by dissolving potassium hydroxide in ethanol. Dissolve the potassium hydroxide in ethanol and make it freshly prepared or keep it well-preserved in a tightly stoppered bottle to avoid absorption of CO₂. Fit a reflux condenser to the flask and reflux the mixture for 30 minutes by gentle boiling to allow complete saponification of the sample. Remove the flask from the heat after refluxing and add immediately 1 to 2 drops of phenolphthalein indicator. Titrate the excess KOH from the hot mixture with standard 0.5 N hydrochloric acid (HCl) solution until the pink colour just disappears, marking the endpoint. Perform a blank titration with the same amount of alcoholic KOH but omitting the sample of plant extract under the same conditions. The difference in the volumes of HCl used in blank and sample titration is used to determine the saponification value. The value is in terms of milligrams of KOH used to saponify 1 gram of the plant extract Sansevieria and calculated from the formula.

Formula:

Saponification value: (A-B)×N × 56.11/W

The saponification value of the water extract of Sansevieria roxburghiana is calculated and results as 93.42mgKOH/g.

In-Vivo Methods:

Acute toxicity studies: The aqueous extract of Sansevieria roxburghiana was given intraperitoneally to albino mice at dosages of 100mg/kg and 200mg/kg by OECD guideline 423. For 14 days, the animals were monitored for any indications of toxicity or death. Since no negative effects were seen, the extract can be safely used at these dosages for additional in vivo analgesic testing.

Animal Grouping: Albino Mice weighing between 20-25g were grouped into four groups, six animals in each group.

Group I served as the control normal saline. Group II animals received 10mg/kg Diclofenac intraperitoneally. Group III as a low dose of sansevieria roxburghiana leaves aqueous extract (SRAE) of 100mg/kg, and group IV as a high dose of sansevieria roxburghiana leaves aqueous extract of 200mg/kg.

Eddy's Hot Plate:

Principle: The point is that nociceptive stimuli, namely thermal stimulation (radiant heat is employed as a painful stimulus), chemical stimulation, and mechanical stimulation, can induce painful responses in experimental animals. Whether animals respond to pain has been tested by the hot plate test. The analysis of the reaction to the pain caused by heat allowed its usage during the research of basic pain principles and in the identification of the efficiency of pain killers. It was useful in basic pain studies and in assessing the efficacy of analgesics through observing the response to heat pain. They utilised a nociception behaviour paradigm where a noxious thermal stimulus provokes an observer behaviour such as jumping and licking of the hind paw. Licking was an obvious indicator of a nociceptive threshold that happened in the form of a quick response in response to unpleasant heat stimuli. A more complicated reaction is that of jumping, where there are the element of a latency and an emotional process of running away¹³.

Procedure: The present study aimed at measuring the central analgesic action of the water extract of Sansevieria roxburghiana using, Eddy hot plate test, which is a proven methodology that measures the response to heat nociceptive stimuli. The test can be used particularly to screen centrally acting analgesics, that is, the drugs which increase the pain threshold by interfering with the central nervous system. The experimental animals (most frequently Swiss albino mice, weighing 20-30g (mice) or Albino mice. These were acclimatised to regular laboratory conditions (12-hour cycle of light and dark, temperature: 22 2^oC, and free access to Galton standard pellet diet and water). The animals were starved on the previous night with a water supply. The animals were arbitrarily separated into 4 random groups consisting of 6 animals each. Group 1 was the vehicle and received normal saline (vehicle) applied intraperitoneally (intraperitoneal route.) Group 2 was the control group, and it received diclofenac sodium 10mg/kg intraperitoneally, a commonly used non-steroidal anti-inflammatory drug (NSAID) which has analgesic and anti-inflammatory action. Group 3 was administered with the aqueous extract of Sansevieria roxburghiana (low dose 100mg/kg i.p.). Group 4, the sansevieria roxburghiana plant leaves aqueous extract was given in a high dose (200mg/kg i.p.). Pain was measured using the Eddy hot plate device. The temperature of the hot plate was adjusted to 55+/-1°C. One animal at a time was put onto the hot plate, and the response was measured, i.e. latency was measured to respond to the heat stimulus (e.g. licking the paw, jumping, withdrawal of the paw). 15-second cut-off time was applied in order to prevent injury to animals. The baseline reaction times were observed, exposing the subject to the drug when the drug had not yet been added (0 minutes) and thereafter, reaction times were taken at 0 min, 30min, 60min, 90 minutes after addition. Considerable rise in reaction time after treatment in comparison with the baseline and control group was considered as a pointer of central analgesic action. The statistical test was done through appropriate statistics, such as ANOVA, followed by post hoc tests to determine the significance of groups. In the current study, it is a practical method to measure the centrally mediated analgesic potential of the drug, and the comparative effect of the plant extract to the standard drug (diclofenac) would give a clue about the possible mechanism of action. In case of an elevation of the threshold of pain being observed when using plant extract, this could have been due to the effect of centrally acting phytoconstituents, which could be of useful application in the treatment of pain.

Fig.6 Weighing of Albino Mice

Fig.7 Administering the drug compound

Fig.8 Placing Albino mice on a Hot plate

Fig.9 Paw licking and jumping responses

Acetic Acid Induced Writhing Test:

Principle:

Writhing test Acetic acid-induced writhing test is a common test used to assess the peripheral analgesic effect of pharmacological agents in animals (usually in mice). The logic behind this test is that pain is endured when exogenous acetic acid is added intraperitoneally and that localised inflammation results in the production of endogenous chemical pain mediators that include prostaglandins (mainly PGE2 and PGF2a), bradykinin, histamine, and serotonin. These chemical mediators cause a sensitisation of the nociceptors to cause a classic constrictive abdominal response that is defined as a writhing response, which involves hind limb stretch, abdominal contractions, and trunk twisting. The severity and extent of such writhing manoeuvres equally correlate to the severity of the pain that the animal is undergoing. The drug analgesics, more so those acting peripherally, as in the use of NSAIDs, will minimise the number of writhes by affecting the formation or activity of these inflammatory mediators. A marked decrease in the number of writhes after administration of the drug as compared to the control group, is an indication of analgesic activity. This model is said to be sensitive, shocking to manipulable, and the advantage is that it detects minimal analgesia. Although this response to peripheral analgesic effects in principle, one can learn a little about central mechanisms, so long as appropriate controls and comparisons are made. This method is, however, subject to a variety of parameters like the concentration of acetic acid, strain of animal, and the conditions of the experiment, which must be standardised very carefully. Generally, the acetic acid writhing test is a fast and easy preliminary test that can be used in the testing of compounds with most likely analgesic activity, particularly those which are active in prostaglandin modes that overcome the pain transmission of the periphery.

Procedure:

In attempting to find out the peripheral analgesic activity of Sansevieria roxburghiana water extract, the test was carried out using acetic acid-induced writhing. This is a regular screening system of assessment of a substance that has the ability to cause reduction of pain through acting peripherally, particularly by inhibition of inflammatory mediators like prostaglandins. In our experiment, the healthy adult laboratory mice were randomly assigned to a group of 4, but the numbers in the three groups were equal (normally 5-6 animals in a group). The velocity of all treatments was done through the intraperitoneal (i.p.). Group 1 receives normal saline as a control. Group 2 was kept as the reference and was given diclofenac sodium (10mg/kg i.p.), which is a common analgesic NSAID, being the reference of peripheral analgesic action. Group 3 received a SRAE low dose (100mg/kg i.p.) of aqueous extract of Sansevieria roxburghiana, whereas Group 4 received a SRAE high dose (200mg/kg i.p.) of the same extract. Writhing in each of the mice was induced 30minutes after administration of the test extract or standard drug by intraperitoneal injection of acetic acid (0.1%) solution (10mL/kg body weight). The mice were then each introduced into transparent observation chambers, and the number of writhing reactions (indicated by the constriction of the abdomen, extension of the hind limbs, and extension of the body) of each mouse was counted within 20 to 30minutes.

The reduction in the number of writhes in the treated groups compared to the control group was considered to show an analgesic effect. The percentage inhibition of writhing was calculated using the formula:

% Inhibition = [(Writhes in control - Writhes in test)/Writhes in control] × 100

The model test is sensitive to steroidal and non-steroidal anti-inflammatory compounds, and an appreciable reduction in writhing number of the groups treated with extracts would signify the presence of a bioactive substance of Sansevieria roxburghiana with peripheral analgesic pain-killing action. Statistical analyses of the data were conducted using appropriate methods of analysis, including one-way ANOVA and post hoc tests, to determine the disparities among groups.

Fig.10 Administration of compounds to albino mice

Fig.11 Placing albino mice in a glass chamber.

Fig. 12 Visible abdominal writhings in albino mice for acetic acid induced writhing test.

RESULTS AND DISCUSSION:

The pain-relieving (analgesic) activity of Sansevieria roxburghiana aqueous extract (SRAE), isolated by Soxhlet extraction, was determined in albino mice by the two developed laboratory animal models, Eddy Hot Plate (HPT) and Acetic Acid-Induced Writhing (AHAT). The animals were selected into four categories, which include control (normal saline), standard (10mg/kg diclofenac sodium), and two test groups in which the SRAE was administered in 100mg/kg and 200mg/kg the administration of drugs through the intraperitoneal route. In the writings about the Eddy Hot Plate assay, there was a progressive elevation of the reaction time in the two groups, which were treated with extracts, but the high dose group achieved a heightened value of latency as compared to the low dose group and controls. The efficacy of the standard drug in terms of alleviating pain was the greatest, as would be anticipated. These results indicate that SRAE could perform its effect via central processes to raise the pain threshold. In the Acetic Acid-Induced Writhing test, the number of writhes decreased with dose in extract-treated groups, and the large dose provided a bigger decrease in the number of writhes than of the small one. The animals that were treated with diclofenac recorded the highest writhing responses. The findings of the two models confirm the central and peripheral analgesic implications of Sansevieria roxburghiana aqueous extract, which supports the assumption that it can be a source of natural pain-relieving agents.

Acetic Acid Induced Writhing Test:

Sansevieria roxburghiana aqueous extract (SRAE) analgesic effect was studied by performing acetic acid induced writhing experiments on albino mice and the time of measuring the action of this effect was done at 30 and 60 minutes after administration in the peripheral system. Group A, which was the control group, demonstrated the normal response to nociceptive effects of acetic acid (26.17±0.66 at 30 minutes and 21.67±1.63 at 60 minutes). Group B, which received the standard drug Diclofenac (10mg/kg) had a significant inhibition of writhing acts with values of 13.16±1.88 and 9.60± 0.86 at the corresponding time points with percentage inhibition of 49.70% and 55.69% respectively. SRAE had a dose-dependent analgesic effect. The group that received a low dose of drug (100mg/kg; Group C) exhibited moderate analgesic effect with the result of 20.13±1.49 (23.11% inhibition) at 30 minutes and 18.45±1.37 (14.88% inhibition) at 60 minutes. As can be observed, the high dose group 200mg/kg (Group D) was more effective with the numbers of writhes recorded at 30 and 60 minutes, reducing to 17.11±1.26 and 13.00 ±1.03, respectively, as well as percent inhibition of 34.61% and 40.00%, respectively. These results show that Sansevieria roxburghiana exerts dose-related peripheral analgesic activities, which are probably due to the regulation of the inflammatory mediator, that is, prostaglandin-like in the acetic acid-induced writhing experiment. The findings confirm the possible value of SRAE as a peripheral acting analgesic agent. (Table-1)

Eddy’s Hot Plate Method:

The main analgesic effect of Sansevieria roxburghiana aqueous extract was measured by the Eddy Hot Plate test, and comparison was made between the control, standard and the extract-test groups. There was a stable control group latency (14.00±0.26 at 0 min to 11.17± 0.39 at 90 min). The analgesic activity of diclofenac (10mg/kg) was also potent, as shown by the percentage latency reduction at 90 minutes, which was 28.79%, 32.59%, 37.32%, and 46.28% at 0, 30, 60, and 90 minutes, respectively. The low dose of S. roxburghiana extract (100mg/kg) produced linearly mild effects of analgesia: 12.33±0.49, 11.93, 10.50±0.59, 11.24%, 9.67±0.28, 10.71%, and 9.33±0.42, 16.47%. The high dose (200mg/kg) elicited dramatic latency enhancement that reached 10.57±0.20(24.50%) at 0min, which was followed by 7.33±0.28 (29.04%) at 30min, 7.17±0.17 s (33.80%) at 60min, at 90 mins 8.39±0.43 is (37.33%). It is a dose-dependent interaction where the high-dose extract is shown to have a strong central analgesic property, nearly similar to the most common drug. It is speculated that bioactive phytoconstituents in S. roxburghiana could have effects on nociceptive through a centric pathway. (Table-2).

Fig.14 The graph representing the data related to the result of Sansevieria roxburghiana aqueous leaves extract (SRAE) using Eddy’s hot method following ANOVA.

The graphical presentation represents the in vivo pain-killing effect of Sansevieria roxburghiana aqueous extract (SRAE) evaluated by acute antinociceptive effect of the mice-based model, tested by monitoring the occurrence of nociceptive responses, i.e., paw licking and jumping behaviour in the mice over 90minutes. We used four experimental groups that included a control group (which was given normal saline), a standard group (which was given Diclofenac sodium (10mg/kg) and two other groups (that were given SRAE at 100mg/kg and 200mg/kg, respectively). The control group recorded the highest noci scores throughout the measurements, which indicates that it did not receive an analgesic treatment. Conversely, pain responses decreased significantly and gradually in the standard group, and it is worth noting that Diclofenac, as one of the reference NSAIDs, was effective. It is interesting to note that the SRAE 100 mg/kg treated animals showed a slight but detectable reduction in paw licking and jumping, which indicated a minimal but small analgesia. In the higher dose group (200mg/kg) the inhibition of nociceptive behaviours was more evident, especially at proposed time points, that is, 60 minutes and 90 minutes, values resembled those achieved with the standard drug group. This indicates the dose-dependent analgesic effect of Sansevieria roxburghiana extract and proves to be pharmacological evidence of its ethnomedicinal importance. The findings point to the hypothesis that SRAE, and particularly its higher concentrations, could have strong central and/or peripheral analgesic properties, which in turn should be studied further regarding its bioactive components included in it and the way they can act.

CONCLUSION:

The present study sheds light on the high pharmacological potential of Sansevieria roxburghiana aqueous extract of leaves, which is acquired by the Soxhlet method, quantified using established in vivo models and can be proved by the physicochemical characterisation. The extract was tested using four experimental groups, which include control, standard (diclofenac 10mg/kg), SRAE low dose (100mg/kg) and SRAE high dose (200mg/kg) treatment in albino mice. The extract contained dose-dependent antinociceptive activity, based on the analgesic activity of the high dose of the extract, which expressed the same degree of analgesic capacity as the standard diclofenac group in both Eddy Hot Plate and Acetic Acid-Induced Writhing assays. Physicochemical composition, such as specific gravity and saponification value, was within acceptable limits of quality products, advocating consistency and stability of the aqueous extract. The pharmacodynamic pattern observed indicates the existence of bioactive phytoconstituents, which can modulate peripheral and central mechanisms of pain. On balance, the results justify the folk application of S. roxburghiana in pain treatment and encourage any subsequent mechanistic or phytochemical study to explain the treatment opportunity in analgesia.

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Received on 16.12.2025 Revised on 15.01.2026

Accepted on 09.02.2026 Published on 22.04.2026

Available online from April 24, 2026

Res.J. Pharmacology and Pharmacodynamics.2026;18(2):131-139.

DOI: 10.52711/2321-5836.2026.00018

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Time	Control (Mean±SEM)	Diclofenac (Mean±SEM)	Diclofenac (% Inhibition)	Low Dose (Mean±SEM)	Low Dose (% Inhibition)	High Dose (Mean±SEM)	High Dose (% Inhibition)
0MINS	14.00±0.26	9.97±0.19**	28.79%	12.33 ± 0.49 *	11.93%	10.57±0.20**	24.50%
30MINS	11.83±0.60	7.98±0.40*	32.59%	10.50 ± 0.59*	11.24%	8.39±0.43*	29.04%
60MINS	10.83±0.34	6.79±0.38**	37.32%	9.67 ± 0.28*	10.71%	7.17±0.17**	33.80%
90MINS	11.17±0.39	6.35±0.34**	46.28%	9.33 ± 0.42*	16.47%	7.00±0.51**	37.33%