Guizhi Fuling Capsule Exhibits Antidysmenorrhea Activity by Inhibition of Cyclooxygenase Activity

Dried materials of Cassia Twig, Indian Bread, Peach Seed, White Peony Root, and Tree Peony Bark were purchased from Hexin Tang Raw Chinese Herbal Medicine Factory. The identities of the materials were authenticated by a botanist from Kanion Pharmaceuticals and validated by HPLC profiling by following protocols given in Chinese Pharmacopoeia (2015 Edition) or by comparison with fingerprints kept at Kanion. Representative voucher specimens were cataloged and deposited at Kanion.

GZFLc were manufactured at Kanion Pharmaceuticals using a formula essentially the same as described in Jingui Yaolue. An equal amount of Cassia Twig, Indian Bread, Peach Seed, White Peony Root, and Tree Peony Bark was used in manufacturing the capsule using a proprietary method developed at Kanion. Each capsule, weighing at 310 mg, is equal to 1.2 grams of herbal materials. The preparation was profiled by HPLC analysis (Figure 1 represents a typical HPLC histogram of GZFLc). The contents of the major components per capsule (310 mg) of the batch used for this study were determined as amygdalin at 6.22 mg, paeonol at 4.43 mg, paeoniflorin at 6.97 mg, and cinnamaldehyde at 0.60 mg.

To prepare a plant extract from individual herbs, raw materials were grounded to pass an 80-mesh screen, weighed, and mixed with distilled water (a gram of powder with 2 ml distilled water) in sealed conical tubes. The contents were extracted twice in a 95°C water bath, each time for 60 min with occasional shaking and vortexing. The extracts were pooled and then filtered through two layers of cheesecloth. After adjusting the volume to approximately 300 mg of raw material/ml (about 30 mg raw material/mouse), freshly prepared extracts were tested for their antidysmenorrhea activity (as summarized in Table 1).

Acetonitrile of HPLC grade was purchased from TEDIA (Fairfield, OH), and analytical reagent grade trifluoroacetic acid was purchased from Nanjing Chemical Reagent Company (Nanjing, China). Ultrapure water was prepared in the lab using a Millipore water purification system (Milford, MA). Paeoniflorin (PubChem ID 442534, purity >98%) and amygdalin (PubChem ID 656516, purity >98%) were purchased from MedChem Express, and paeonol (PubChem ID 24895579, purity >99%) and cinnamaldehyde (PubChem ID 24900954, purity >95%) were purchased from Sigma Aldrich (Shanghai, China).

Antibodies against COX2 and β-actin were purchased from Santa Cruz. HRP-conjugated secondary antibodies were purchased from Sigma Aldrich. The ECL reagent kit was purchased from Thermo Fisher. A COX2 inhibitor screening kit was purchased from Beyotime Technologies (#S0168, Nantong, China).

HPLC studies were carried out using a Waters 6890 liquid chromatograph system. The separation was carried out on a Kromasil reverse phase C18 column (Kromasil KR100-5-C18, 4.6 ∗ 250, 5 μm) using a gradient consisting of 0.02% (v/v) trifluoroacetic acid in water (TFA) as mobile phase A and acetonitrile as mobile phase B. A linear gradient program was set as follows: 0–5 min, 95% A, 5–20 min, 95–83% A, 20–30 min, 83–81% A, 30–40 min, 81–74% A, 40–60 min, 74–12% A, and 60–70 min of 12% A. The column temperature was maintained at 30°C, while the flow rate was kept constant at 1.0 ml per min.

To profile the chemicals in the plants, grounded materials were accurately weighed and extracted in a 37°C water bath with methanol and analyzed. The identity of the major components was determined by comparison with corresponding standards (as summarized in Table 1).

The animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of Medical School of Nanjing University and by the IACUC of Kanion Pharmaceutical Co., respectively. The animal care and experimental procedures were carried out in accordance with the Guidelines for the Care and Use of Laboratory Animals set by the US NIH and Regulations for the Care and Use of Laboratory Animals (2008) set by Jiangsu Province of China.

Female ICR mice (6–8 weeks old) were purchased from Nanjing University model animal facility. The animals were housed under standard laboratory conditions, and food and tap water were provided ad libitum. For animal studies, ibuprofen or the contents (30 mg/ml) of GZFLc were resuspended in a PBS buffer containing 10% DMSO and 2% Tween 80 and administrated without storage by oral gavage.

We used an induced-dysmenorrhea model essentially as described [16]. Briefly, ICR mice were treated with saline or with estradiol benzoate (1 mg/kg/day) by i.p. route for three consecutive days. On the fourth day, the mice were randomly grouped (n = 5 or 10) and were treated by oral gavage with saline (n = 5 for untreated control; n = 10 for oxytocin-induced, but untreated), or with ibuprofen (50 mg/kg, n = 10), or a test sample (n = 10) at dosages as indicated. The animals were then injected with 50 U/kg of oxytocin (MedChem Express, Shanghai) by i.p. injection 30 min after drug administration. Oxytocin injection resulted in immediate writhing responses, which mainly consist of abdominal wall contractions and pelvic rotation, followed by hind limb stretches. The responses were recorded for 30 min.

We adapted a rotarod method to determine the therapeutic effect by measuring the time of a mouse staying on a rotating rod [17, 18]. After administration of oxytocin, mock- or drug-treated mice, five in a group (n = 5), were put on a rotarod apparatus (model YLS-4C, Zhongshidi Technologies, Beijing) with the rotating rate set at 20–30 rpm. The time that a mouse dropped off from the rotating rod was recorded and used to measure the severity of dysmenorrhea.

For histological or immune blotting studies, the animals were sacrificed by cervical dislocation 20 min after oxytocin treatment. The uterine tissues were collected and fixed in 10% buffered formalin or processed for protein extraction and immune blotting studies. Fixed organs were dehydrated with a graded ethanol series and embedded in paraffin sectioning and prepared for hematoxylin and eosin staining. The slices were examined under a light microscope (Olympus BX53 microscope). The images were captured with the equipped camera and preinstalled software.

The uterine tissues taken after writhing study were used to prepare total protein extract. Tissues, two from each group, were lysed with a lysis buffer containing 1% NP-40 and 0.1% SDS and a cocktail of protease inhibitors. The samples were left on ice for 30 min with occasional vortexing. The tissue lysates were collected after centrifugation at 10,000  ×g for 10 min at 4°C. After determining protein concentrations using a BCA method, the proteins were separated by SDS and immune-blotted with an appropriate antibody and an ECL reagent kit. The images were captured on Chem Scope equipment.

The effect of GZFLc or pure chemicals on COX2 activity was measured using cyclooxygenase-2 inhibitor screening kit (#S0168). The kit measures the conversion of arachidonic acid to prostaglandin G2 by COX2. Briefly, recombinant human COX2 in 96-well plates was incubated at 37°C for 15 min with series-diluted GZFLc or a test compound in duplicate. COX2 specific inhibitor celecoxib was included as a positive control. The reaction mixtures were then allowed to react with arachidonic acid following the manufacturer's instruction. COX2 activity was determined by measuring the production of the final product with a fluorescent probe. The excitation wavelength was set at 560 nm and the emission wavelength was at 590 nm. The inhibitory effect on COX2 was calculated using the following equation: percentage of inhibition = (1 − average net inhibitor fluorescence/average net noninhibited fluorescence) × 100%.

The data were expressed as average ± SE or mean ± SD. One-way ANOVA was used for comparison of statistic significances among paired groups.

We and others have shown that GZFLc or similar formulae were able to reduce pains and ameliorate symptoms associated with dysmenorrhea. To delineate the major ingredients responsible for GZFLc effect, the contents of GZFLc were resuspended in PBS and tested in a mouse dysmenorrhea model developed by Yang et al. [16]. In this regard, forty-five ICR female mice were treated with estradiol for three consecutive days. On the fourth day, the animals were randomly grouped into five groups (Table 2) and were treated with ibuprofen (50 mg/kg, orally) or with GZFLc at 150 and 300 mg/kg, respectively. We chose those two dosages since they fall within the recommended ranges for human usage. Thirty minutes after the treatment, the animals were injected by i.p. route with oxytocin at 50 U/kg or with saline for the mice in the control group. Oxytocin injection induced significant but momentary pain in mice that lasted about 20 min, including signs of abdominal wall contractions, pelvic rotation, and rear limb stretches. As shown in Table 2, mice treated with ibuprofen had significantly less symptoms of writhing responses. For comparison, GZFLc at 150 and 300 mg/kg markedly reduced the frequency of pain responses, particularly those of pelvic twisting and rear limb stretches, from 20 times to 7 and 4 times, respectively, indicating that GZFLc was effective against oxytocin-induced writhing responses. A comparison between the two groups was made. As shown in Table 3, except that there was no difference between the control group and ibuprofen group, there was a significant difference between the other two groups (p < 0.05).

Dysmenorrhea causes pain that might affect motor coordination and balance of the animals. We attempted to use a rotarod assay to quantitatively measure the effect of GZFLc against dysmenorrhea. In this regard, randomly grouped mice (n = 5) were subjected to a test by putting the animals on a rotating bar. The time that an animal stays on the rotating bar was recorded and used to measure the effect of ibuprofen or GZFLc treatment. As shown in Figure 2(a), the average time of mice hanging on the bar was recorded as 12.95 ± 2.68 min, while the time dropped to less than 2 min in the oxytocin-induced group. Treatment with ibuprofen brought the hanging time close to that of the nonoxytocin-induced controls (12.63 ± 2.07 min). Treatment with 150 and 300 mg/kg GZFLc resulted in the hanging time to be 8.74 and 10.12 min, respectively, indicating that GZFLc treatment alleviated the discomfort of mice suffering from dysmenorrhea.

The antidysmenorrhea effect was further investigated by examination for signs of proinflammatory responses. In this study, mice were killed 20 min after oxytocin treatment. The uterine tissues were removed and fixed for H & E staining (Figure 2(b)). Compared with the uteruses from the control group (Con), the lamina propria had severe edema and the basalis layer of endometrium was thicker, while the endometrium had more endometrial glands in estradiol-treated and oxytocin-induced group (Mock), indicating that the animals were in the estrus stage. The lamina propria in ibuprofen (Ibup) and in GZFL-treated groups had markedly reduced edema compared with oxytocin-induced and vehicle-treated controls (Mock group), while no obvious differences were noticed in the basalis layers and the endometrium among Mock and GZFLc-treated groups.

The results together demonstrated that GZFLc treatment was effective against induced dysmenorrhea.

Prostaglandin F2α production has been identified as a critical factor for primary dysmenorrhea [6, 10, 19]. We therefore examined whether GZFLc treatment affected COX2 expression, a rate determining enzyme that catalyzes arachidonic acid conversion to prostaglandins. The mouse uterus tissues from untreated or ibuprofen or GZFLc-treated samples were used for COX2 expression studies. As shown in Figure 3(a), COX2 expression was relatively low in the untreated samples, while its expression was significantly upregulated in estradiol and oxytocin-treated samples. Unlike results from those with prolonged treatment, we detected no significant alteration of COX2 expression in GZFLc or in ibuprofen-treated samples, indicating GZFLc treatment did not change COX2 expression.

To preliminarily determine the contributions of individual components, grounded plant materials were extracted at 95°C for 60 min in sealed tubes. The extracts were cleansed by filtering through two layers of cheesecloth and were reconstituted to match the contents in GZFL formula. The extracts were then tested individually on the oxytocin-induced dysmenorrhea model. Although the effect of individual extracts was less potent compared to that of 300 mg/kg of GZFLc, the extracts from radices Cassia Twig, White Peony Root, and Tree Peony Bark, but not Indian Bread and Peach Seed, showed activities in reducing writhing responses (Figure 4). In general, extracts from Cassia Twig, White Peony Root, and Tree Peony Bark reduced the frequencies of pelvic twisting and limb stretches by approximately 60–70%, indicating that those materials were partially responsible for the antidysmenorrhea activity of GZFLc.

Based on the results from activity-directed assay, we investigated whether the main components were representative of GZFLc activity. The chemical contents in GZFLc have previously been identified, with cinnamaldehyde, paeoniflorin, paeonol, and amygdalin as major components [20, 21]. We therefore profiled the chemicals from Cassia Twig, White Peony Root, and Tree Peony Bark by HPLC analysis since extracts from those plants showed activity against oxytocin-induced writhing responses. Consistent with previous studies, HPLC profiling indeed showed paeoniflorin, paeonol, and cinnamaldehyde as the major representative compounds in those plants (Figure 5). We therefore tested whether those major components represented GZFLc activity. Paeoniflorin at 30 μM partially blocked COX2 activity, while paeonol and cinnamaldehyde showed marginal to no activity in the same enzyme assay (Figure 6(a)). We also tested whether those compounds blocked oxytocin-induced dysmenorrhea. We used dosages of 5 and 10 mg/kg for paeoniflorin (0.5 mg/ml) and paeonol (0.5 mg/ml) and 1 and 2 mg/kg for cinnamaldehyde (0.1 mg/ml) in animal studies since those dosages would fall close to the ranges of the compounds in the capsule (refer to Section 2). We found that administration of paeoniflorin and paeonol, but not cinnamaldehyde, reduced the numbers of limb stretches and pelvic twisting (Figure 6(b), upper panels). The treatment also prolonged the time of mice staying on a rotating bar (Figure 6(b), lower panel). The effect was more profound for paeoniflorin and paeonol (Figure 6(b)). In addition to showing that these compounds represent GZFLc activity against oxytocin-induced dysmenorrhea, the results suggested to us that inhibition of COX2 in part represents GZFLc antidysmenorrhea activity.