INTRODUCTION:

Inflammation is an emergency reaction that shows signs of redness, heat, swelling and pain resulting from infections caused by pathogenic organisms, toxins, chemical and physical stimuli, irradiation, bruises and caustic agents^1-3. The inflammation process is a physiological response to trauma aimed at minimizing impacts that would have been known to be otherwise detrimental⁴. The inflammation process starts with chemical mediators derived from macrophages and neutrophil cells involved in initiating, progression, regulating and termination of acute inflammation. If this strength is not set in the quick phase there will be a chronic phase as pointed out above⁵. Lately, chronic inflammatory disease has been thought of as the leading killer, being responsible for more than over 50 percent of all fatalities worldwide caused by ischemic cardiac problems, chronic renal disease, diabetes, neurodegenerative as well as autoimmune related disorders⁶. The two broad categories of inflammation-inhibiting constitute steroidal agents and non-steroidal agents. The non-steroidal compounds are employed to relieve slight to moderate pain and as anti-febrifuge due to cyclooxygenase enzyme blockade⁷. However, they have several side effects, which include cardiovascular risks as well as damaging irritation in the stomach⁸. Therefore, a systematic review of the different species of plants and products containing active compounds was undertaken since they might harbor new compounds with anti-inflammatory effects, fewer side effects and are cheaper⁹. It has been found that pain is defined a as sensation ranging from moderate to severe soreness. Pain can be localized and specific like in case there was an injury and can be not localized at all. Sparing use of analgesic drugs is usually taken when a patient is in that stage where they are having moderate pain and are using drugs to control such pain^1,10. As per the World Health Organisation, It is predicted that 80% citizens of the world's, continue to use plant-derived drugs and this includes the remedial utilization of plants as anti-nociceptive drugs used traditionally^11-14.

The renowned fruit tree known as the wild apricot (Prunus armeniaca Linn.) can be found in the country's mid-hills and arid temperate zones. The wild apricot is part of the Prunoideae subfamily of the Rosaceae family. In the Kumaon region of Uttarakhand, wild apricot is found in Nainital, Almora, and Pithoragarh districts. P. armeniaca L. contains many phytochemicals like sugars, carbohydrates, polyphenols, fatty acids, sterol compounds, carotenoids, cyanogenic-type glucosides, and volatile components. Traditionally, the apricot’s different parts are employed in the management of several illnesses like cough, asthma, bronchitis, anemia, and fever and are also used as food additives¹⁵. Numerous polyphenolic substances found in apricot root have an essential character in the management and treatment of degenerative illnesses like cancer and cardiovascular conditions. Proanthocyanidins, also known as condensed tannins, belong to the class flavonols, and are one of them. Proanthocyanidins are phenolic compounds and possess antioxidant, analgesic, anti-inflammatory, anticarcinogenic, and antimicrobial activities. It has been discovered in earlier investigations that proanthocyanidins inhibit platelet aggregation, lipid peroxidation, vascular permeability, as well as fragility¹⁶. The primary active component in apricot roots has been found to be proanthocyanidins having doubly-linked structures. Ephedrannin A and a novel A-type proanthocyanidin-entepiafzelechin-3-oxy-parahydroxybenzoate(4-8,2-O-7)-epiafzelechin were discovered in the roots of P. armeniaca L. in earlier studies¹⁷. Therefore, based on the multiple mechanisms and activity reported by proanthocyanidins, this work was performed to assess the inflammation-suppressing, analgesic, and antioxidant functions of P. armeniaca L. roots hydroalcoholic extract as there are no studies have been reported on this topic in the medical history.

METHODS AND MATERIAL:

Collection and Validation of Plant Matter:

In the months of April and September 23, P. armeniaca roots were collected from the villages of Dugtanidhar and Satbuna village, Ramgarh (Mukteshwar), district Nainital, Uttarakhand, India. At the Botanical Survey of India, Dehradun, Uttarakhand, their authentication took place.

Plant Extracts Preparation:

The apricot roots were first coarsely crushed and air-dried to prepare the plant matter. The plant matter was macerated with ethanol and separated after being dried and ground into powder. Then filter paper (Whatman) was used to filter it. Utilising a rotatory evaporator, the produced extract was subjected to concentration and then dried at a lower temperature. For later usage, the extract was refrigerated in an airtight bottle at 4 degrees Centigrade.

Chemicals and Drugs:

Normal saline (0.9%), butylated hydroxyl toluene (BHT), 1,1 diphenyl-2-picryl hydrazyl (DPPH), Folin-Ciocaleteau reagent, ascorbic acid, potassium ferricyanide, sodium nitroprusside, pyrocatechol, methanol, trichloroacetic acid, sulphanilamide, carrageenan (1%), ethanol, sulphuric acid, distilled water, vanillin reagent, ferric salt, n-butanol, Mayer’s, Wagner’s, Hager’s, Legal, Molish’s, Benedict’s, Fehling’s reagents and standard drugs (diclofenac sodium, indomethacin), etc. were incorporated into the study.

Equipments:

Eddy’s hot plate, plethysmometer, rotatory evaporator, UV spectrophotometer, stopwatch, water bath and other tools were applied in this research.

Qualitative Evaluation of Phytochemicals of Plants:

The preliminary phytochemical activity was performed to find out the existence of active ingredients in the hydroalcoholic extract of P. armeniaca roots. The appearance of condensed tannins (proanthocyanidins) was examined by performing vanillin and acid butanol tests^18-25.

In-Vitro Examination of Antioxidant Activity:

Radical Scavenging Potential Using the DPPH Method:

The 1,1 diphenyl-2-picryl hydrazyl (DPPH) procedure was taken to assess the ability to quench reactive oxygen species by ARE²¹. For this, some tubes of root extract were made using ethanol (70%) at varying concentrations (50, 75, 100, 200, and 300 g/ml). By adding ethanol (70%), the samples' volume as well as of the ascorbic acid went up to 3 ml. Each test tube received 1ml of the methyl alcohol mixture of DPPH (100 M or 0.1mM), which was then violently shaken and allowed to stand at 27 degrees Centigrade for 30min. Without using any samples or the standard (ascorbic acid), a control sample was produced using the above-mentioned approach. 3ml of 70% ethanol and methanol (1ml) were mixed to create the benchmark. The absorbance was noted at 517nm. All of the aforementioned methods were run three times, and their average was then calculated. A higher level of free radical scavenging activity is reflected by a reaction mixture's reduced absorbance^26-29.The formula used to determine the % scavenging is: Percentage scavenging = [(A₀– A₁)/A₀] ×100, Here, the absorbances related to the control group and extract/standard, respectively, are marked by A₀ and A₁. The IC₅₀ parameter was determined by graphing the quantity of extract (μg/ml) against the percentage reduction using an equation of a straight line^30-32.

Radical Scavenging Activity Using Nitric Oxide:

In this method, firstly the experimental mixture (3 ml) was made by adding a 2ml aliquot of 5mM sodium nitroprusside with phosphate-buffered saline and 1ml of extract in a range of amount (50, 75, 100, 200, and 300 g/ml with DMSO (di-methyl sulphoxide)). Following that, this composition was held for incubation at 25 degrees centigrade for 30min. After the reaction mixture had been incubated, 1.5ml of the test sample was collected, and 1.5ml of the Griess reactant was included. Chromophore was generated when nitrite ions were subjected to diazotization with sulphanilamide and were then mixed with naphthyl ethylene diamine. This chromophore's absorbance was measured at 546nm^33-38. There was no test or standard sample taken for the control sample. Ascorbic acid was taken as the standard compound. A total of three attempts for each test were carried out before averaging the results. Through a comparison of the test and control sample’s absorbance, the percentage reduction of nitric oxide (NO) production was measured is: % Radical scavenging activity = [(A_control – A_sample / A_control)] × 100. Wherein A_sampledisplays the absorbance of the test substance and A_controldisplays the absorbance of the control. By plugging the numbers from the graph of sample concentration vs percentage inhibition of free radicals into a regression equation, the IC50 values—the concentrations of samples that scavenge 50% of free radicals were computed.

Reductive Ability:

The apricot root extract's ability to reduce was assessed using Oyaizu's method³⁹. Extract and standard solution were made in methanol, at different volumes (50, 75, 100, 200, and 300g/ml). We took 1ml of this solution and combined it with 2.5ml aliquot of potassium ferricyanide (1% in sterile water) and 2.5 ml of buffered phosphate solution having pH 6.6. Following that, The mixture was left to incubate for 20 minutes at 50 degrees Centigrade. After that, the mixture was processed using 2.5ml of trichloroacetic acid (10% in distilled water) and left for ten minutes of centrifugation at 3000rpm. After that, 2.5ml of distilled water and 0.5ml of ferric chloride were mixed in the solution's topmost layer (2.5ml). Next, the solution’s absorbance was noted at a wavelength of 700nm. BHT served as the reference substance. The outcomes were averaged after each analysis was carried out in triplicate. The increased absorbance of the resultant solution reflects its high reducing power^40,41.

Assessment of Total Phenol Concentration:

Applying the Folin-Ciocalteu reagent, the quantity of total phenolic compounds in the ARE was determined, and the results were represented as pyrocatechol equivalents⁴². This was accomplished by dissolving ARE in an aqueous methanol solution (6:4 v/v) that had been prepared. Following that, multiple concentrations of the standard drug pyrocatechol (10, 20, 40, 60, 80, and 100g/ml) and single amount (100g/ml) of the ARE were collected in dissimilar laboratory tubes and distilled water was included to get the amount up to 0.2 ml. To each tube, 0.8ml of a 7.5% sodium carbonate mixture and 1ml of the Folin-Ciocalteu reagent were put on. At 765nm, the solution's absorbance was assessed after keeping the solution for 30 minutes in incubation. Following the completion of each experiment in triplicate, the findings were averaged. Using a formula derived from a typical pyrocatechol graph, the total phenolic components in the concentrate were estimated as µg of pyrocatechol equivalent. The equation is: Absorbance = 0.014 × pyrocatechol (μg) + 2.444

Metal Chelating Activity:

0.1mM FeSO4 (0.2ml) as well as 0.25mM ferrozine (0.4 ml) solutions were successively introduced into 0.2ml of ARE to measure the metal chelating activity⁴³. The solution's absorbance was then determined at 562 nm after keeping it for 1 minute at moderate temperature. As the conventional medication, ethylenediamine trichloroacetic acid (EDTA) was employed. The equation was used to measure metal chelating activity is: Metal chelating activity = [(A_control – A_sample / A_control)] × 100, Where A_control denotesthe absorbance of the control sample (excluding apricot root extract) while A_sample denotes the absorbance of the plant concentrate.

In-Vivo Activities:

Animals:

Both sexes of Swiss albino mice having weight approx. 25 and 30g have been taken from the animal section of the Kumaun University Nainital, Uttarakhand, India for the experiments. All of the animals were placed in disinfected cages of polypropylene and kept in a heat-monitored environment (22°C+3°C) with 12/12hour light-dark cycles. The Institutional Animal Ethics Committee (IAEC) provided its approval to all experimental techniques and protocols utilized in the study, all CPCSEA regulations were adhered to.

Acute Oral Toxicity Study:

According to OECD (Organisation for Economic Co-operation and Development) guideline 423 (acute toxic class approach), oral acute toxicity research was conducted⁴⁴. For this, albino Wistar rats that were in good health (i.e., nulliparous and not pregnant) were used. ARE was given orally in amounts up to 2000 mg/kg. Following that, all experimental animals were checked for any indication of poisonous effect, illness, or fatality for the initial 24 hr and followed by the next 72hr.

Evaluation of Analgesic Activity:

Analgesic Activity by Employing Hot Plate Method:

Six mice were placed in each of the four groups of animals. Animals in Group I received vehicle (0.5ml distilled water/kg body weight, p.o.), Group II got standard medication (10mg of diclofenac sodium, p.o.), and Groups III and IV received two dissimilar doses of the ARE (200 and 400mg, p.o., respectively). Then, animals from each group were placed on Eddy's heating plate, which was retained at a constant 55 degrees centigrade. To stop paw injury, a closing time of 15 seconds was taken into consideration. Reaction times were measured for each group at 0, 30, 60, 90, and 120 minutes after taking samples when animals licked or jumped^45,46.

Central Analgesic Activity Evaluation by Tail Immersion Assay:

The same groups of animals were divided in this approach as in the one described previously. The baseline latency was assessed at intermittent times of 0, 30, 60, 90, and 120 min, before and after drug treatment. Each mouse's tail tip (1-2cm) was immersed in a thermostatically regulated water bath at standard temperature (45±1 degree centigrade) after the administration of the intended medication. Stopwatch measurements of the mice's actual flick responses were made, and the outcomes were compared in contrast to those of the standard and control groups. To prevent harming the tissues of the tails, the maximum time limit for immersion was 180 s^47,48,36.

Evaluation of Anti-Inflammatory Activity:

Analysis of Anti-Inflammatory Potential by Employing Carrageenan-trigerred Hind Paw Edema Method:

The carrageenan technique was employed to examine the anti-inflammatory potential of apricot root extract^49,50.By injecting 0.1ml of carrageenan mixture (1% w/v in normal saline) in the right back paws of the mice, acute inflammation was produced using this method. All of the mice were separated into 5 groups for this study: one group served as a negative control, two groups received varying amounts of root extract, and two more groups served as the control and standard. The negative control group got a carrageenan mixture (1% w/v in normal saline, delivered by injection), on the other hand, the control group merely got vehicle (0.5ml distilled water/kg body weight, p.o.). The control group received indomethacin (10mg/kg, body mass p.o.). Extracts were administered in two distinct doses (200 and 400mg/kg, body mass, p.o., respectively) 60 minutes before the injection of carrageenan. Digital plethysmometers were used to measure paw volume at intervals of 0, 30, 60, and 90, after carrageenan injection⁵¹.

Statistical Analysis:

The results of the experimental measurements reported in study are given as mean = (number±standard deviation). The quantitative analysis of mean parameters between the groups applied in the current study was done by one-way analysis of variance (ANOVA). In all analyses, the p value was accepted as p ≤ 0.05. All the figures in this research were generated using Graph Pad Prism software⁵².

RESULTS:

Results of Phytochemical Screening:

Chemical tests were conducted, and the outcomes are listed in table 1. Results showed that ARE had a significant amount of condensed (proanthocyanidins) tannins, as well as carbohydrates, alkaloids, proteins, amino acids, glycosides, and terpenoids.

Table 1: Results of the different chemical tests performed

S. No.	Chemical constituents	Tests	Results
1	Carbohydrates	Molisch’s, Barfoed’s, Fehling’s and Benedict’s tests	+
2	Proteins	Millon’s test	+
3	Amino acids	Biuret and Ninhydrin tests	-
4	Steroids	Salkowski test, Liebermann-Burchard tests	+
5	Flavonoids	Sulphuric acid and Lead acetate tests	+
6	Alkaloids	Mayer’s, Dragendorff’s, Hager’s and Wagner’s tests	+
7	Tannins and Phenolic compounds	5% Ferric Chloride test;	+
7	Tannins and Phenolic compounds	Acetic acid solution test	-
8	Saponins	Foam/Froth test	+
9	Glycosides	Keller–Killiani test	+
9	Glycosides	Legal test	-
10	Proanthocyanidins (condensed tannins)	Acid butanol and Vanillin Tests	+

+: Positive result; -: Negative result

In-Vitro Antioxidant Activity:

The hydroalcoholic extract of P. armeniaca L. was chosen in five concentrations ranging from 50 to 300 g/ml, and its antioxidant potential was assessed using several in-vitro techniques. It was discovered that test samples at various concentrations quenched free radicals. All antioxidant models (DPPH, nitric oxide radicals, and metal chelating activities) had maximal inhibitory concentrations (IC50) that were discovered to be 91.44, 81.11, and 169.8g/ml, respectively. Further analysis revealed that the total polyphenol content was 16.79mg GAE/g of dry material with good lowering ability. Figures 1 to 8 depict the percentage scavenging of apricot root extract and standard medications at various doses.

Acute Toxicity Study:

When administered orally, the maximum dose of the hydroalcoholic extract of P. armeniaca L. roots (ARE) (2000mg/kg) din’t show any acute toxic reactions. Additionally, mortality was not found for the 24-hour observation period. Thus, ARE was discovered to be safe at doses of 2 gm/kg body weight orally in mice, so two doses (200 and 400mg/kg) were finalized to determine the in-vivo pain relieving and inflammation suppression activities.

Figure 1. DPPH quenching activity of apricot root extract (RE) compared with standard ascorbic acid (AA)

Figure 2. Graphical representation of scavenging activity of ARE against DPPH radical generation, where ARE= Apricot root extract

Figure 3. Nitric oxide radical quenching activity of apricot root extract (RE) compared with standard ascorbic acid (AA)

Figure 4. Graphical representation of scavenging activity of ARE against NO radical generation, where ARE= Apricot root extract, AA= Ascorbic acid

Figure 5. Metal quenching activity of apricot root extract (RE) compared with standard ethylene diamine tetra acetic acid (EDTA)

Figure 6. Graphical representation of metal ion chelating activity of ARE, where ARE=root extract, EDTA= Ethylene diamine tetraacetic acid

Figure 7. Standard curve of pyrocatechol for determination of total phenolic content of apricot root

Figure 8. Reductive ability of different concentrations of apricot root extract (RE) and standard drug butylated hydroxy toluene (BHT)

Analgesic Activity:

Hot Plate Method:

The P. armeniaca L. root extract produced concentration-dependent analgesic efficacy. The outcomes for the hot plate method are manifested in tables 2-3 and figures 9-10. By using the paw-licking method, ARE at the doses of 200 mg/kg and 400 mg/kg showed a significant effect (*P<0.05 and **P<0.01) in contrast to the control group. However, using the paw jumping method, ARE had a significant effect (**P<0.01, ***P<0.001) when compared to the animals of control group at the same doses. The strongest analgesic activity was measured having dose of 400 mg/kg after 120 mins by both paw licking and paw jumping methods.

Table 4: Analgesic potential of P. Armeniaca L. Root Extract by Tail Immersion Model

Groups	Drug/Extract	Dose	0 mins	30 mins	60 mins	90 mins	120 mins
I	Control	Distilled water (0.5 ml/kg)	2.2±0.056	2.51±0.085	2.38±0.258	2.43±0.326	2.46±0.187
II	Diclofenac Sodium	10 mg/kg p.o.	3.35±0.099	4.4±0.135***	5.4±0.073***	6.65±0.088***	7.3±0.126***
III	Apricot root extract	200 mg/kg p.o.	2.4±0.073	3.18±0.079*	3.5±0.112*	4.15±0.092*	4.25±0.056*
IV	Apricot root extract	400 mg/kg p.o.	2.9±0.057	3.38±0.090**	4.3±0.106**	5.33±0.098**	6.06±0.076**

*P < 0.05, **P < 0.01, ***P < 0.001 vs. control, (data is presented as mean ± SEM from each group's six animals)

Table 5: Anti-inflammatory Potential of P. Armenica Root Extract Using Carrageenan-Stimulated Paw Edema Method in Mice

Groups	Drug/Extract	Dose	0 mins	30 mins	60 mins	90 mins	120 mins
I	Control	Distilled Water (0.5 ml/kg) 1% w/v in normal saline	0.5516± 0.0149	0.596± 0.0234	0.751± 0.0349	0.738± 0.0312	0.78± 0.0332
II	Carrageenan	(0.5 ml/kg) 1% w/v in normal saline	1.12± 0.029**	1.16± 0.025***	1.28± 0.011***	1.38± 0.021***	1.60± 0.018***
III	Indomethacin	10 mg/kg p.o.	0.94±0.008**	0.81±0.01**	0.66±0.019**	0.43±0.011**	0.29±0.01**
IV	Apricot root extract	200 mg/kg p.o.	1.12±0.022**	0.93±0.015*	0.82±0.019*	0.81±0.025*	0.705±0.012*
V	Apricot root extract	400 mg/kg p.o.	1.12±0.023**	0.80±0.034**	0.71±0.015**	0.65±0.023**	0.55±0.014**

Data are given as mean ± SEM (n=6), one-way ANOVA and Dunnett test; *P < 0.05, **P < 0.01, in contrast to the carrageenan group and^¨¨¨P < 0.001, ^¨¨P < 0.01 in compare with the control group.

Figure 12. Effect of apricot root extract on carrageenan-induced paw edema in mice. Data are exhibited as mean ± SEM (n=6), one way ANOVA is followed by Dunnett test; *P < 0.05, **P < 0.01, in contrast to the carrageenan group. ^¨¨P < 0.01, ^¨¨¨P < 0.001 when carrageenan group compared with control group

DISCUSSION AND CONCLUSIONS:

Two distinct techniques hot plate along tail immersion were used in the current investigation to assess the hydroalcoholic extract of P. armeniaca L.'s analgesic efficacy. A carrageenan-elected paw edema technique was employed to assess the anti-inflammatory potential. We measured both activities at 200 and 400 mg/kg doses. For analgesic and anti-inflammatory potential, standard medications included indomethacin and diclofenac sodium. By extending the reaction time of mice in opposed to the control group, ARE provided effective analgesic activity in the hot plate and tail immersion techniques. Higher ARE concentrations have produced better results. By lengthening the reaction time of mice's tail withdrawal activity, opiates, and opiate-like medications mimic all of the steps in the tail immersion method. As one of the finest ways to test anti-inflammatory medicines, the suppression of inflammation caused by carrageenan was used to evaluate anti-inflammatory activity. The anti-oedematous activity of the medication has frequently been evaluated by employing the carrageenan-triggered paw edema model. We discovered that ARE (200 and 400mg/kg) demonstrated considerable dose-dependent inhibition against carrageenan-elicted paw swelling. In this technique, ARE also showed effective peripheral anti-inflammatory action. Thus the hydroalcoholic extract of P. armeniaca L. root produced notable anti-oxidant, antinociceptive, and anti-inflammatory effects in a concentration-dependent way, it was determined. According to a study, flavonoids prevent the production of several prostaglandin-producing enzymes such as prostaglandin synthetase and endoperoxidase. Because prostaglandins cause pain and inflammation, inhibiting their synthesis is probably the cause of ARE's analgesic and anti-inflammatory effects. Because prostaglandins cause pain and inflammation, inhibiting their synthesis is probably the cause of ARE's antinociceptive and anti-inflammatory potential. Due to their significant antioxidant effects and suppression of prostaglandin formation, the discovered proanthocyanidins may also be accountable for the hydroalcoholic extract's antioxidant, analgesic, and anti-inflammatory properties⁵³. To determine whether the apricot plant is entirely effective, more investigation is needed to determine any vital components that may be present. This research can be used to develop and synthesize different dosage forms using the apricot root's active molecules, which are thought to provide antioxidant, analgesic, and anti-inflammatory action. The development and following of such a natural remedy will play a principal role henceforth.

CONFLICT OF INTEREST:

No conflicts of interest.

ACKNOWLEDGMENT:

I am grateful to Dr. Archana N. Sah from the depth of my heart for her valuable and helpful guidance in the execution of this study. I am thankful for her willingness to part with her valuable time in such a thoughtful manner.

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Received on 06.05.2025 Revised on 09.09.2025

Accepted on 03.11.2025 Published on 01.07.2026

Available online from July 04, 2026

Research J. Pharmacy and Technology. 2026;19(7):2917-2925.

DOI: 10.52711/0974-360X.2026.00416

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

Groups	Drug Extract	Dose	0 mins	30 mins	60 mins	90 mins	120 mins
I	Distilled water	0.5 ml/kg	4.8±0.2215	4.28±0.1195	6.157±0.2943	5.11±0.2908	5.655±0.2257
II	Diclofenac sodium	10 mg/ kg p.o.	5.959± 0.1719	9.7± 0.2165**	11.55± 0.1893**	13.72± 0.2939**	15.05± 0.2303**
III	Apricot root extract	200 mg/ kg p.o.	6.123± 0.09462	7.162± 0.13*	9.743± 0.152*	11.13± 0.2966*	11.97± 0.1958*
IV	Apricot root extract	400 mg/ kg p.o.	6.007± 0.3188	7.95± 0.1599*	9.282± 0.1404*	12.09± 0.2342*	12.649± 0.3288*

Groups	Drug/Extract	Dose	0 mins	30 mins	60 mins	90 mins	120 mins
I	Distilled water	0.5 ml/kg	7.83±0.181	7.45±0.175	6.691±0.219	7.795±0.195	7.04±0.277
II	Diclofenac Sodium	10 mg/ kg p.o.	11.1±0.230	13.52± 0.220***	13.63± 0.151***	14.6± 0.221***	14.17± 0.418***
III	Apricot root extract	200 mg/ kg p.o.	8.08±0.187	10.17± 0.190**	12.0± 0.163**	12.07± 0.295**	13.006± 0.320**
IV	Apricot root extract	400 mg/ kg p.o.	11.69±0.351	11.88± 0.150***	12.65± 0.278***	13.73± 0.482***	14.42± 0.247***