PerioH-035 is composed of the radix of Angelica sinensis, radix of steamed Rehmannia glutinosa, radix of Angelica dahurica, rhizome of Cimicifuga heracleifolia, and fruit of Zanthoxylum piperitum. Each 100 g of dried herb underwent extraction with 1000 mL 50% ethanol for 24 hours at room temperature. The extracts of each herb were filtered, evaporated, and lyophilized. A freeze-dryer was used to dry the extracts of each herb. The powders obtained were stored at -20℃ until use (Angelica sinensis 37.33 g, steamed Rehmannia glutinosa 50.12 g, Angelica dahurica 34.55 g, Cimicifuga heracleifolia 15.03 g, and Zanthoxylum piperitum 5.61 g). The final yields of each herb were 37.33%, 50.12%, 34.55%, 15.03%, and 5.61%, respectively. A voucher specimen (PD- PerioH-035) was deposited at our laboratory.

Male Sprague-Dawley rats aged 7 weeks (RaonBio Inc., Yongin, Korea) were housed in standard conditions (12 h light/dark cycle, 20±2℃ temperature and 50±5% humidity) with access to food and water ad libitum. All experiments were conducted according to the guidelines of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Care and Use of Laboratory Animals of Kyung Hee University (KHUASP(SE)-14-029).

The rats were randomly assigned to 1 of 4 groups (n = 7, respectively): NL (non-ligatured), L (ligatured), P1 (ligatured and treated with 1 mg/mL PerioH-035), P100 (ligatured and treated with 100 mg/mL PerioH-035). The sample size was determined based on previous studies[1920]. After adaptation for 1 week, a sterilized 3-0 nylon thread ligature was placed around the cervix of the bilateral mandibular first molars of rats under anesthesia with intraperitoneal injection of a tiletamine/zolazepam mixture (Zoletil 50; Virbac Lab, Carros, France), except in the NL group. For the treatment, 20 mg of 5 herbal powders were mixed and dissolved in 1 mL distilled water (100 mg/mL of PerioH-035). To enhance the viscosity, 1% carboxymethyl cellulose was added to the samples. The normal group, NL, received no treatment and the negative control group, L, was treated with vehicle gel. The PerioH-035-treated groups, P1 and P100, were topically treated with 100 µL of 1 and 100 mg/mL PerioH-035 on the bilateral first molars. The treatments were continued once per day for 2 weeks.

The left mandibles were analyzed by micro-computed tomography (micro-CT; SkyScan, SCANCO USA, Southeastern, PA, USA), with a minor modification of a previously published protocol after excising the ligatures [21]. Using three-dimensional image reconstructions, the cementoenamel junction (CEJ) and marginal bone crest were drawn manually. Alveolar bone resorption was measured as the distance from the CEJ to the alveolar bone crest.

The right mandibular molar regions were retrieved following the excision of the ligatures and fixed in 10% neutralized formalin for 18 hours. De-fleshed molar samples were stained with 1% aqueous methylene blue (Sigma, MO, USA) for 5 minutes. Digital photography was obtained from a 100 mm macro lens Canon digital camera (Canon, Tokyo, Japan). The root axis of the first molar teeth (three roots) was measured with a computerized densitometry system Image J (NIH, Bethesda, MD, USA). The amount of the alveolar bone loss (ABL) was the average of the values of the measurements from three points along the CEJ to the alveolar bone crest.

Following the micro-CT, the left mandibular molar regions were separated and fixed in 10% neutralized formalin for 18 hours. To decalcify, 0.1 M ethylene diamine tetraacetic acid aqueous solution was used for 2 months at room temperature with general shaking. The decalcified molars were embedded in paraffin. 7-µm cut sections were stained with hematoxylin and eosin (H&E). The digital images were acquired using Leica Application Suite (LAS) microscope software (Leica Microsystems, Buffalo Grove, IL, USA). The magnifications used were 40× and 200×.

The 7-µm cut sections were stained with tartrate-resistant acid phosphatase (TRAP; Sigma, St. Louis, MO, USA) according to the manufacturer’s instruction. Naphthol AS-BI phosphate was used as a substrate. The TRAP-stained slides were again stained with hematoxylin to identify the multinuclear cells. The digital images were acquired using Leica Application Suite (LAS) microscope software. The magnification used was 200×.

Total RNA from the detached gingival tissues was isolated with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. A volume of 1 µg of the total RNA from each sample was isolated and synthesized into cDNA using commercially available cDNA synthesis kits (Invitrogen Corp., Carlsbad, CA, USA) at 45℃ for 60 minutes and then at 95℃ for 5 minutes. The samples were then stored at -20℃ until further use. Amplification of cDNA was conducted with Taq polymerase and primers specific for matrix metalloproteinase (MMP)-9 mRNA and GAPDH. The following primers were used: MMP-9, 5′-GGG ACG CAG ACA TCG TCA TC-3′ (forward) and 5′-TCG TCA TCG TCG AAA TGG GC′ (reverse); and GAPDH, 5′-CCA TCA CCA TCT TCC AGG AG -3′ (forward) and 5′-CCT GCT TCA CCA CCT TCT TG-3′ (reverse). Optimum conditions for RT-PCR were established using routine methods. The conditions used for MMP-9 were 35 cycles of 94℃ for 20 seconds, 60℃ for 30 seconds and 72℃ for 2 minutes, with final extension at 72℃ for 10 minutes. The PCR products were electrophoresed in a 1% agarose gel. Relative mRNA expression was quantified with Quantity One software using the GAPDH mRNA expression level as a reference. Visualized bands were quantified using a computerized densitometry system, Image J.

Significance was determined by one-way analysis of variance (ANOVA) and Dunnett’s multiple comparison tests. In all analyses, P<0.05 was taken to indicate statistical significance.

Three-dimensional reconstruction from micro-CT images revealed the ABL, and the amount of exposed root surface was about 1.52 times higher in the L group, which was significant (NL = 785.67±208.5 µm; L = 1,197.07±234.22 µm; P<0.001), and the treatment of PerioH-035 100 mg/mL significantly inhibited the alveolar bone resorption by up to 15.30% (1,014.04±221.81 µm; P<0.05) (Fig. 1). However, there was no statistical difference between L and P1.

Similar results were shown in the methylene blue staining. The rats with ligatures showed significant ABL—about 1.32 times that of NL (NL = 2,720±130 µm; L = 3,610±280 µm; P<0.001). It was observed that treatment with PerioH-035 1 and 100 mg/mL (3,003±140 and 2,920±310 µm; 16.81% and 19.11%, respectively) significantly reversed the ABL caused by ligature-induced periodontitis (Fig. 2).

Multinuclear cells, which appeared TRAP-positive cells, were markedly increased by ligature induction as shown in the L group. Increased TRAP-positive cells were reduced by the treatment with PerioH-035, especially in the P100 group (Fig. 3).

In the ligature-induced periodontitis model, severe inflammatory infiltration of the gingival tissues were observed compared with the NL group and exhibited loss of attachment, severe bone resorption, apical migration of junctional epithelium, and cementum demineralization. Several resorption pits were also observed on the cementum surface. However, the treatment with 100 mg/mL PerioH-035 appeared to prevent the destruction of periodontal tissues and the connective attachment was maintained over the cementum lining along the root surface (Fig. 4).

The expression of MMP-9 mRNA of gingival tissue in the L group was significantly higher than that in the NL group (NL = 1±0.08; L = 14.1±0.11; P<0.05). However, topical application of PerioH-035 significantly decreased the expression of MMP-9 mRNA in gingival tissues at all concentrations compared with the L group (P1 = 8.7±0.29; P100 = 7.3±0.34; P<0.05) (Fig. 5).