Enhancement effect of ultrasonification process on UV-protection and skin-whitening activities using Centella asiatica L. Urban extract was investigated. Cytotoxicity of the extracts measured on human skin fibroblast cells, CCD-986sk, and then, ultrasonification associated extracts showed 5~9% lower cytotoxicity then normal crude extracts on 1.0 mg/ml of highest sample concentration. The associated extrats showed highest inhibition activity of hyaluronidase on 1.0 mg/ml of concentration as 54.2%. Also, the associated extract reduced expression of MMP-1 on UV-irradiated CCD-986sk cells down to 100.2% from 136.1%, and revealed high inhibitory potency on tyrosinase as 74.6% by adding 1.0 mg/ml of concentration. Ultrasonification associated extract showed strong inhibition effect of melanin production on Clone M-3 cells as 84.2% by adding 1.0 mg/ml of concentration. From the preliminary observations, we considered that the extracts from C. asiatica could be potent natural materials for skin-whitening and anti-aging agent, and could enhance the activities by ultrasonification process.

Phosphatidylcholine was used to encapsulate aqueous extracts of Centella asiatica, and its biological activity was compared with another aqueous extracts. Nanoparticle of C. asiatica was made by encapsulation to w/o type spherical liposome which of aqueous extracts seized with oil phase as 78.2 nm average diameter. Cytotoxicity of the nanoparticle was measured on human skin fibroblast cells, CCD-986sk, and showed lower cytotoxicity on 1.0 mg/ml of highest concentration as 28% than that of another extracts. The nanoparticle showed the highest promotion of human B and T cell growth up to 138% and 135%, respectively, compared to the control. and the NK cell growth was promoted up to 8% higher than the control in proportion to secretion of IL-6 and TNF-α from immune cell growth. Also nanoparticle showed highest inhibition activity of hyaluronidase on 1.0 mg/ml of highest concentration as 60.5%. It seems that because of enhanced biological application of aqueous extracts on cell through nano-encapsulation process.

본 연구는 병풀의 초음파 추출 시 용매에 따른 면역활성 증진 효과를 탐색하고자 수행되었다, 각 추출공정의 수율 비교에서 초음파 공정을 병행한 60℃ 에탄올 추출이 일반 100℃ 물 추출에 비해 15% 높은 수율을 나타냈으며, 에탄올을 용매로 한 추출이 동일한 조건의 물 추출에 비해 모두 높은 수율을 나타냈다. 이는 용매의 극성과 용출되는 병풀 내 성분의 극성에 기인하는 것으로 사료된다. 인간 정상 섬유아세포인 CCD-986sk를 이용한 세포독성 측정을 통해 모든 병풀 추출물이 32% 이하의 세포독성을 나타냈다. 인간 면역 B, T세포의 생육도 측정에서 초음파를 병행한 60℃ 에탄올 추출물이 무첨가 대조구에 비해 10%의 증진 효과를 나타내었으며, 면역세포의 cytokine 분비량 측정에서도 IL-6, TNF-α의 분비를 각각 4.86 × 10-4 pg/cell과 5.73 × 10-4 pg/cell로 나타내 증진 효과를 나타냈다. 또한, 초음파 병행 에탄올 시료를 첨가한 면역 T세포의 분비물에 의한 NK 세포 활성에서도 10%의 생육증진효과를 나타내었다. 이를 통해 병풀의 에탄올 추출물이 면역 활성을 가지고 있으며 초음파 공정을 통해 활성의 증진이 나타날 수 있음을 확인하였다. 이는 에탄올 용매를 통해 병풀 유용성분의 용출이 증진되고, 초음파 공정을 통해 병풀 유용 성분의 수율 향상 및 유용성분의 변화, 신규물질 용출 등이 일어나기 때문인 것으로 사료된다.

Objectives Here, we report the effect of overexpression of ginseng farnesyl diphosphate synthase on the transcription of three key regulatory enzymes involved in triterpene metabolism in hairy root of ginseng and Centella asiatica (L.) Urban. Materials and Methods A four-year-old root of Panax ginseng C.A. Meyer and Centella asiatica (L.) Urban whole plants were obtained from National Institute of Crop Science (Suwon, Korea) and Chonnam National University (Gwangju, Korea), respectively. Agrobacterium rhizogenes R1000 strain was kindly provided by Dr. In (Nongwoo Bio, Yeju, Korea). Results and Discussion The role of farnesyl diphosphate synthase (FPS) in triterpene biosynthesis (Fig. 1) was investigated. A pCAMBIA3101 vector was used to insert a exogenous gene into target plant genome (Fig. 2). After the transformation, we produced Panax ginseng and Centella asiatica hairy roots by introducing the coding region of the gene from Panax ginseng. In these hairy roots, integration of the transgenes into the C. asiatica nuclear genome was confirmed by PCR analysis using PgFPS (P. ginseng FPS) primers and by Southern hybridization using PgFPS-specific probe. FPS specific activity is increased 4-fold compared to controls. In RT-PCR analysis, overexpression of PgFPS in hairy roots was observed (Fig. 3) and two genes, cycloartenol and beta-amyrin synthase, related to triterpene biosynthesis were up-regulated. These results suggest that FPS overexpression might lead to an enhanced biosynthesis of triterpene saponins and phytosterols. However, we did not demonstrate whether or not the introduction of PgFPS gene in Centella asiatica genome directly enhances triterpene saponin production, although our results showed that gene expression related to triterpene saponin biosynthesis were obviously up-regulated. Therefore, additional experiments such as overexpression of FPS gene in triterpene saponin-deficient mutant plants will be required.

An efficient plant regeneration of C. asiatica was achieved from organogenesis using petiole explants of in vitro plantlet on MS basal medium controled with different plant growth regulators (NAA,2,4-D, IAA kinetin, and BA). Best results that 50%, efficiency of regeneration per explant for regeneration were obtained with IAA 17.13 μM and BA 8.9 μM. Formation of adventitious shoots via organogenesis from the petiole explant was verified by histological sectioning of plantlets. Regenerated plants were transplanted into soil.

Centella asiatica accumulates large amounts of triterpene saponin, such as centellasaponin, asiaticoside, madecassoside. We examined the effect of two candidates, MJ (Methyl jasmonate) and TDZ (thidiazuron), on asiaticoside production and the accumulation of bAS mRNA associated with asiaticoside biosynthesis in leaves of cultured whole plants. The growth of whole plants treated with 0.1 mM MJ was found to decrease significantly, however, the growth of whole plants treated with 0.1 mM MJ plus 0.025 mg/l TDZ was better than that treated with MJ alone. When MJ alone was added to culture medium, asiaticoside contents in leaves were higher than that of control after 7 days of treatments. The maximum level of bAS (β-amyrin synthsae) mRNA in leaves of whole plant treated TDZ and MJ was transiently observed after exposure to 5 days. These results showed the up-regulation of bAS gene by adding TDZ and MJ at the molecular level, however, synergic effects of TDZ and MJ on asiaticoside biosynthesis were not testified.

Plants have been known to accumulate a very diverse range of triterpene saponins. We have investigated the regulation of saponin biosynthesis in higher plants using Centella asiatica (L.) Urban as a model plant. Effects of a feeding precursor on asiaticoside production from leaves and on the level of two-type OSCs mRNA were investigated. As a feeding precursor, squalene negatively affected the levels of CYS and bAS mRNA, but it also decreased the production of asiaticoside from whole plants. Plant hormones regulate secondary metabolism, and in plant tissue cultures they could affect both culture growth and secondary metabolite production. Although enhancement of asiaticoside production from whole plant cultures by addition of TDZ (thidiazuron) has been reported, the positive effect of TDZ on the levels of OSCs transcripts was not observed.

5월부터 10월까지 병풀의 지상부에 triterpene glycosides인 madecassoside와 asiaticoside의 시기별 함량변화를 조사하였다. 잎에서는 9월에 약 108.1 mg/g으로 가장 높은 수준의 함량을 보였다. 엽병에서는 19.02 mg/g으로 잎에서 보이는 수준보다는 낮은 수준을 나타냈다. 병풀의 triterpene glycosides는 잎에 주로 축적되며, 가장 적절한 병풀의 수확시기는 9월로 잠적 확인되었다.