Rg3-enriched red ginseng extracts enhance apoptosis in CoCl 2 -stimulated breast cancer cells by suppressing autophagy

Yun-Jeong Jeong,Mi-Hee Yu,Yuna Cho,Min-Young Jo,Kwon-Ho Song,Yung Hyun Choi,Taeg Kyu Kwon,Jong-Young Kwak,Young-Chae Chang 고려인삼학회 2024 Journal of Ginseng Research Vol.48 No.1

Background: Ginsenoside Rg3, a primary bioactive component of red ginseng, has anti-cancer effects. However, the effects of Rg3-enriched ginseng extract (Rg3RGE) on apoptosis and autophagy in breastcancer have not yet been investigated. In the present study, we explored the anti-tumor effects ofRg3RGE on breast cancer cells stimulated CoCl2, a mimetic of the chronic hypoxic response, and determinedthe operative mechanisms of action. Methods: The inhibitory mechanisms of Rg3RGE on breast cancer cells, such as apoptosis, autophagy andROS levels, were detected both in vitro. To determine the anti-cancer effects of Rg3RGE in vivo, the cancerxenograft model was used. Results: Rg3RGE suppressed CoCl2-induced spheroid formation and cell viability in 3D culture of breastcancer cells. Rg3RGE promoted apoptosis by increasing cleaved caspase 3 and cleaved PARP anddecreasing Bcl2 under the hypoxia mimetic conditions. Further, we identified that Rg3RGE promotedapoptosis by inhibiting lysosomal degradation of autophagosome contents in CoCl2-induced autophagy. We further identified that Rg3RGE-induced apoptotic cell death and autophagy inhibition was mediatedby increased intracellular ROS levels. Similarly, in the in vivo xenograft model, Rg3RGE induced apoptosisand inhibited cell proliferation and autophagy. Conclusion: Rg3RGE-stimulated ROS production promotes apoptosis and inhibits protective autophagyunder hypoxic conditions. Autophagosome accumulation is critical to the apoptotic effects of Rg3RGE. The in vivo findings also demonstrate that Rg3RGE inhibits breast cancer cell growth, suggesting thatRg3RGE has potential as potential as a putative breast cancer therapeutic

Antihyperlipidemic Effect of Ginsenoside Rg1 in Type 2 Diabetic Mice

Jae Hong Park(박재홍),Jiyoun Lee(이지연),Jiyoung Yeo(여지영),Jeong Su Nam(남정수),Myeong Ho Jung(정명호) 한국생명과학회 2011 생명과학회지 Vol.21 No.7

Ginsenoside Rg1은 인삼에서 분리한 약물학적인 활성을 가지는 물질이다. 본 연구는 Rg1이 제2형 당뇨병 모델 동물에서 혈당과 지질대사에 유익한 효과를 가지는지를 확인하기 위한 목적으로 수행되었다. 10주령의 db/db 마우스에 Rg1을 10 ㎎/㎏ 농도로 15일간 경구투여한 결과 공복혈당이 감소하였고, 포도당 내성이 개선되었다. 특히 혈중 중성지방과 유리지방산이 유의적으로 감소하였고 혈중 HDL-콜레스테롤이 증가되었다. 또한 chimeric GAL4-PPARα receptor 활성 프로모터를 활성화시켰고 PPARα gene인 CPT-1 (carnitine palmitoyltransferase-1)과 ACO (acyl-CoA oxidase)의 발현을 증가시켰는데 이것으로 Rg1의 지질대사 개선이 PPARα 활성에 의한 지방산 산화에 의한 것임을 확인할 수 있었다. 모든 결과를 종합해 볼 때, Rg1은 제2형 당뇨병과 관련된 고혈당증과 고지혈증에 유용한 효과를 가짐을 확인하였다. Ginsenoside Rg1 is a pharmacologically active component isolated from ginseng. The goal of this study was to clarify the beneficial effects of Rg1 on glucose and lipid metabolism in diabetic animals (db/db mice). To accomplish this, ten week old db/db mice were administered 10 ㎎/㎏ of Rg1 for 15 days. Rg1 did not influence the weight of db/db mice when compared with vehicle-treated db/db mice. The administration of Rg1 lowered fasting plasma glucose, and improved glucose tolerance. Importantly, Rg1 markedly reduced both plasma triglyceride and free fatty acids, and increased high-density lipoprotein cholesterol (HDL-C) concentrations in db/db mice. Rg1 activated promoter activity of chimeric GAL4-PPARα reporter and increased expression of peroxisome proliferator-activated receptor alpha (PPARα) target genes such as carnitine palmitoyltransferase-1 (CPT-1) and acyl-CoA oxidase (ACO), which are involved in fatty acid oxidation. These findings indicated that improvement of lipid profiles by Rg1 may be associated with increased fatty acid oxidation via PPARα activation. Taken together, these results suggest that Rg1 could have beneficial effects for controlling hyperglycemia and hyperlipidemia associated with type 2 diabetes.

Protective Effects of Ginsenoside Rg₃ against Cholesterol Oxide-Induced Neurotoxicity in the Rat

Kim, Jong-Hoon The Korean Society of Ginseng 2009 Journal of Ginseng Research Vol.33 No.4

Ginsenosides are among the most well-known traditional herbal medicines frequently used for the treatment of various symptoms in South Korea. The neuroprotective effects of ginsenoside $Rg_3$ (G-$Rg_3$) on cholesterol-oxide-(CO)-induced neurotoxicity were investigated through the analyses of rat brains. The recently accumulated reports show that ginseng saponins (GTS), the major active ingredients of Panax ginseng, have protective effects against neurotoxin insults. In the present study, the neuroprotective effects of G-$Rg_3$ on CO-induced hippocampal excitotoxicity were examined in vivo. The in-vitro studies using rat cultured hippocampal neurons revealed that G-$Rg_3$ treatment significantly inhibited CO-induced hippocampal cell death. G-$Rg_3$ treatment not only significantly reduced CO-induced DNA damage but also attenuated CO-induced apoptosis. The in-vivo studies that were conducted revealed that the intracerebroventricular (i.c.v.) pre-administration of G-$Rg_3$ significantly reduced i.c.v. CO-induced hippocampal damage in rats. To examine the mechanisms underlying the in-vitro and in-vivo neuroprotective effects of G-$Rg_3$ against CO-induced hippocampal excitotoxicity, the effect of G-$Rg_3$ on the CO-induced elevations of the apoptotic cells in cultured hippocampal cells was examined, and it was found that G-$Rg_3$ treatment inhibited CO-induced apoptosis. The histopathological evaluation demonstrated that G-$Rg_3$ significantly diminished the apoptosis in the hippocampus and also spared the hippocampal CA1, CA3, and dentate gyrus neurons. G-$Rg_3$ also significantly improved the CO-caused behavioral impairment. G-$Rg_3$ itself had no effect, however, on the CO-induced inhibition of succinate dehydrogenase activity (data not shown). These results collectively indicate the G-$Rg_3$-induced neuroprotection against CO in rat hippocampus. With regard to the wide use of G-$Rg_3$, this agent is potentially beneficial in treating CO-induced brain injury.

ginsenoside Rg3에 의한 B16F10 흑색종 세포의 세포사멸 유도

이슬기(Seul Gi Lee),김병수(Byung Soo Kim),남주옥(Ju-Ock Nam) 한국생명과학회 2014 생명과학회지 Vol.24 No.9

Ginsenoside Rg3는 홍삼으로부터 추출한 활성 성분들 중 하나로 한방 의학에선 원기를 회복시키는 약제로 잘 알려져 있는 인체에 유효한 화학 성분이다. Rg3는 지금까지 많은 연구들에 의하여 다양한 암세포로부터 강력한 항암효과를 가진다고 알려져 있다. 그러나 Rg3가 악성 흑색종 세포에서 어떻게 세포사멸을 유도하는지에 대한 작용 기작은 명백하게 밝혀지지 않았다. 따라서, 본 연구에서는 ginsenoside Rg3가 B16F10 흑색종 세포에서 세포사멸 유도 활성 및 기전에 관한 영향을 조사하였다. 세포 생존력을 MTT assay 법으로 수행한 결과, B16F10 세포에선 농도 의존적으로 세포증식 저해 효과가 나타났고 정상세포인 EA.hy.926 과 NIH3T3 에서는 나타나지 않았다. B1610 세포에 Rg3를 농도 별로 처리 후, TUNEL 염색을 한 결과 세포사멸이 농도 의존적으로 증가 하는 것을 확인 할 수 있었다. Western blot 분석을 실시한 결과, Rg3를 처리한 B16F10 세포에서 p-FAK, Bcl-2, pro-caspase3 단백질들의 발현이 감소 되었고 이와 반대로 Bax, p-p38의 발현은 증가되었다. 따라서, 본 연구에서는 Rg3가 B16F10 흑색종 세포에서 항암제의 agent로써 사용 될 수 있다는 것을 입증하였다. Ginsenoside Rg3 is one of the active ingredients extracted from red ginseng, and it is an effective chemical component of the human body and well known in herbal medicine as a restorative agent. Several studies have shown that Rg3 has a potent anti-tumor effect on various cancer cell lines. However, Rg3-induced apoptosis in B16F10 melanoma cancer cells is not well understood. In the present study, we tested whether ginsenoside Rg3 could induce apoptosis in B16F10 melanoma cells. We found that Rg3 could inhibit B16F10 melanoma cell viability in a dose-dependent manner, but not normal cells, such as EA.hy.926 and NIH3T3 cells. We also found that Rg3 could induce apoptosis in B16F10 melanoma cells using tunnel-staining assay in a dose-dependent manner. Rg3 treatment induces the phosphorylation of p38 and the expression of Bax, but it inhibits the expressions of the phosphorylation of focal adhesion kinase Bcl2 and pro-caspase3. Taken together, our data suggest that Rg3 could be useful as an anti-cancer agent in B16F10 melanoma cells.

Protective Effects of Ginsenoside Rg₃ against Cholesterol Oxide-Induced Neurotoxicity in the Rat

Jong-Hoon Kim 고려인삼학회 2009 Journal of Ginseng Research Vol.33 No.4

Ginsenosides are among the most well-known traditional herbal medicines frequently used for the treatment of various symptoms in South Korea. The neuroprotective effects of ginsenoside Rg₃ (G-Rg₃) on cholesterol-oxide-(CO)-induced neurotoxicity were investigated through the analyses of rat brains. The recently accumulated reports show that ginseng saponins (GTS), the major active ingredients of Panax ginseng, have protective effects against neurotoxin insults. In the present study, the neuroprotective effects of G-Rg₃ on CO-induced hippocampal excitotoxicity were examined in vivo. The in-vitro studies using rat cultured hippocampal neurons revealed that G-Rg₃ treatment significantly inhibited CO-induced hippocampal cell death. G-Rg₃ treatment not only significantly reduced CO-induced DNA damage but also attenuated CO-induced apoptosis. The in-vivo studies that were conducted revealed that the intracerebroventricular (i.c.v.) pre-administration of G-Rg₃ significantly reduced i.c.v. CO-induced hippocampal damage in rats. To examine the mechanisms underlying the in-vitro and in-vivo neuroprotective effects of G-Rg₃ against CO-induced hippocampal excitotoxicity, the effect of G-Rg₃ on the CO-induced elevations of the apoptotic cells in cultured hippocampal cells was examined, and it was found that G-Rg₃ treatment inhibited CO-induced apoptosis. The histopathological evaluation demonstrated that G-Rg₃ significantly diminished the apoptosis in the hippocampus and also spared the hippocampal CA1, CA3, and dentate gyrus neurons. G-Rg₃ also significantly improved the CO-caused behavioral impairment. G-Rg₃ itself had no effect, however, on the CO-induced inhibition of succinate dehydrogenase activity (data not shown). These results collectively indicate the G-Rg₃-induced neuroprotection against CO in rat hippocampus. With regard to the wide use of G-Rg₃, this agent is potentially beneficial in treating CO-induced brain injury.

Protective Effects of Ginsenoside Rg3 against Cholesterol Oxide-Induced Neurotoxicity in the Rat

김종훈 고려인삼학회 2009 Journal of Ginseng Research Vol.33 No.4

Ginsenosides are among the most well-known traditional herbal medicines frequently used for the treatment of various symptoms in South Korea. The neuroprotective effects of ginsenoside Rg3 (G-Rg3) on cholesterol-oxide-(CO)-induced neurotoxicity were investigated through the analyses of rat brains. The recently accumulated reports show that ginseng saponins (GTS), the major active ingredients of Panax ginseng, have protective effects against neurotoxin insults. In the present study, the neuroprotective effects of G-Rg3 on CO-induced hippocampal excitotoxicity were examined in vivo. The in-vitro studies using rat cultured hippocampal neurons revealed that G-Rg3 treatment significantly inhibited CO-induced hippocampal cell death. G-Rg3 treatment not only significantly reduced CO-induced DNA damage but also attenuated CO-induced apoptosis. The in-vivo studies that were conducted revealed that the intracerebroventricular (i.c.v.) pre-administration of G-Rg3 significantly reduced i.c.v. CO-induced hippocampal damage in rats. To examine the mechanisms underlying the in-vitro and in-vivo neuroprotective effects of G-Rg3 against CO-induced hippocampal excitotoxicity, the effect of G-Rg3 on the CO-induced elevations of the apoptotic cells in cultured hippocampal cells was examined, and it was found that G-Rg3 treatment inhibited CO-induced apoptosis. The histopathological evaluation demonstrated that G-Rg3 significantly diminished the apoptosis in the hippocampus and also spared the hippocampal CA1, CA3, and dentate gyrus neurons. G-Rg3 also significantly improved the CO-caused behavioral impairment. G-Rg3 itself had no effect, however, on the CO-induced inhibition of succinate dehydrogenase activity (data not shown). These results collectively indicate the G-Rg3-induced neuroprotection against CO in rat hippocampus. With regard to the wide use of G-Rg3, this agent is potentially beneficial in treating CO-induced brain injury. Ginsenosides are among the most well-known traditional herbal medicines frequently used for the treatment of various symptoms in South Korea. The neuroprotective effects of ginsenoside Rg3 (G-Rg3) on cholesterol-oxide-(CO)-induced neurotoxicity were investigated through the analyses of rat brains. The recently accumulated reports show that ginseng saponins (GTS), the major active ingredients of Panax ginseng, have protective effects against neurotoxin insults. In the present study, the neuroprotective effects of G-Rg3 on CO-induced hippocampal excitotoxicity were examined in vivo. The in-vitro studies using rat cultured hippocampal neurons revealed that G-Rg3 treatment significantly inhibited CO-induced hippocampal cell death. G-Rg3 treatment not only significantly reduced CO-induced DNA damage but also attenuated CO-induced apoptosis. The in-vivo studies that were conducted revealed that the intracerebroventricular (i.c.v.) pre-administration of G-Rg3 significantly reduced i.c.v. CO-induced hippocampal damage in rats. To examine the mechanisms underlying the in-vitro and in-vivo neuroprotective effects of G-Rg3 against CO-induced hippocampal excitotoxicity, the effect of G-Rg3 on the CO-induced elevations of the apoptotic cells in cultured hippocampal cells was examined, and it was found that G-Rg3 treatment inhibited CO-induced apoptosis. The histopathological evaluation demonstrated that G-Rg3 significantly diminished the apoptosis in the hippocampus and also spared the hippocampal CA1, CA3, and dentate gyrus neurons. G-Rg3 also significantly improved the CO-caused behavioral impairment. G-Rg3 itself had no effect, however, on the CO-induced inhibition of succinate dehydrogenase activity (data not shown). These results collectively indicate the G-Rg3-induced neuroprotection against CO in rat hippocampus. With regard to the wide use of G-Rg3, this agent is potentially beneficial in treating CO-induced brain injury.

Neuroprotective Effects of Ginsenoside Rg₃ against 24-OH-cholesterol-induced Cytotoxicity in Cortical Neurons

Yoon Seok Roh,Hyoung Bae Kim,Chang-Won Kang,Bum Seok Kim,Seung-Yeol Nah,Jong-Hoon Kim 고려인삼학회 2010 Journal of Ginseng Research Vol.34 No.3

Ginsenoside Rg₃ (Rg₃), one of the active ingredients in Panax ginseng, attenuates NMDA receptor-mediated currents in vitro and antagonizes NMDA receptors through a glycine modulatory site in rat cultured hippocampal neurons. In the present study, we examined the neuroprotective effects of Rg₃ on 24-hydroxycholesterol (24-OH-chol)-induced cytotoxicity in vitro. The results showed that Rg₃ treatment significantly and dose-dependently inhibited 24-OH-chol-induced cell death in rat cultured cort ical neurons, with an IC?? value of 28.7 ± 7.5 ㎛. Furthermore, the Rg₃ treatment not only significantly reduced DNA damage, but also dose-dependently attenuated 24-OH-chol-induced caspase-3 activity. To study the mechanisms underlying the in vitro neuroprotective effects of Rg₃ against 25-OH-chol-induced cytotoxicity, we also examined the effect of Rg₃ on intracellular Ca²? elevations in cultured neurons and found that Rg₃ treatment dose-dependently inhibited increases in intracellular Ca²?, with an IC?? value of 40.37 ± 12.88 ㎛. Additionally, Rg₃ treatment dose-dependently inhibited apoptosis with an IC?? of 47.3 ± 14.2 ㎛. Finally, after confirming the protective effect of Rg₃ using a terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay, we found that Rg₃ is an active component in ginseng-mediated neuroprotection. These results collectively indicate that Rg₃-induced neuroprotection against 24-OH-chol in rat cortical neurons might be achieved via inhibition of a 24-OH-chol-mediated Ca²? channel. This is the first report to employ cortical neurons to study the neuroprotective effects of Rg₃ against 24-OH-chol. In conclusion, Rg₃ was effective for protecting cells against 24-OH-chol-induced cytotoxicity in rat cortical neurons. This protective ability makes Rg₃ a promising agent in pathologies implicating neurodegeneration such as apoptosis or neuronal cell death.

Ginsenoside Rg1 전환에 대한 에탄올 농도와 유기산의 영향

장귀영(Gwi Yeong Jang),김민영(Min Young Kim),이윤정(Yoon Jeong Lee),이상훈(Sang Hoon Lee),황인국(In Guk Hwang),최재훈(Jehun Choi),신유수(Yu Su Shin),이준수(Junsoo Lee),정헌상(Heon Sang Jeong) 한국식품영양과학회 2018 한국식품영양과학회지 Vol.47 No.8

본 연구에서는 유기산과 에탄올 농도가 인삼의 주요 사포닌인 ginsenoside Rg1의 전환에 미치는 영향을 확인하기 위하여, ginsenoside Rg1 용액에 유기산(citric acid, malic acid 및 succinic acid)과 에탄올 농도를 달리하여 130°C에서 가열 후 Rg1의 변화를 확인하였다. Ginsenoside Rg1은 증류수 조건에서는 130°C에서도 매우 안정하였으며, 유기산이 존재하는 조건에서는 minor ginsenoside로 쉽게 전환되었다. Rg1으로부터 가수분해 반응으로 C-20에 결합한 glucose가 이탈한 Rh1(S) 및 Rh1(R)이 생성되고 가수분해 된 ginsenoside로부터 탈수반응으로 C-20의 OH기가 이탈한 Rk3와 Rh4가 생성되었으며, ginsenoside Rg1의 전환반응은 유기산의 종류와 농도 및 에탄올 농도에 의존적이었다. 또한, Rg1과 Rg1으로부터 생성되는 ginsenoside, 유기산, 에탄올 및 가열시간 간에 유의적인 상관관계를 나타내었다. 이러한 결과는 인삼의 추출용매로서 흔히 사용되는 에탄올이 가열과정에서 ginsenoside의 전환에 많은 영향을 미칠 있으며, 에탄올 농도의 조절이 인삼가공품의 품질지표성분인 ginsenoside 조성을 조절하는 데 활용될 수 있을 것으로 판단된다. This study was performed to determine the influence of ethanol concentration and organic acids on conversion of ginsenoside Rg1 under heating. Ginsenoside Rg1 solution was treated with various ethanol concentrations (0∼50%) and organic acids (0∼3 mM of citric acid, malic acid, and succinic acid) at 130°C, then analyzed using a HPLC-UVD. The ginsenoside Rg1 was highly stable at 130°C in distilled water, whereas Rg1 was readily converted to minor ginsenosides, including Rh1(S), Rh1(R), Rk3, and Rh4 in distilled water with organic acids. The conversion ratio of ginsenosides increased with increasing acid concentration and heating time, whereas it decreased when ethanol concentration increased. The conversion level of Rg1 differed according to types of organic acids. The independent variables (heating time, organic acid, and ethanol concentration) were significantly correlated with ginsenoside conversion (P<0.001). Taken together, these results indicate that the ginsenoside conversion level differed according to ethanol concentration under the same conditions. Therefore, ethanol could be used as a regulator for the conversion of ginsenosides during ginseng extraction and processing.

Neuroprotective Effects of Ginsenoside Rg₃ against 24-OH-cholesterol-induced Cytotoxicity in Cortical Neurons

Roh, Yoon-Seok,Kim, Hyoung-Bae,Kang, Chang-Won,Kim, Bum-Seok,Nah, Seung-Yeol,Kim, Jong-Hoon The Korean Society of Ginseng 2010 Journal of Ginseng Research Vol.34 No.3

Ginsenoside $Rg_3$ ($Rg_3$), one of the active ingredients in Panax ginseng, attenuates NMDA receptor-mediated currents in vitro and antagonizes NMDA receptors through a glycine modulatory site in rat cultured hippocampal neurons. In the present study, we examined the neuroprotective effects of $Rg_3$ on 24-hydroxycholesterol (24-OH-chol)-induced cytotoxicity in vitro. The results showed that $Rg_3$ treatment significantly and dose-dependently inhibited 24-OH-chol-induced cell death in rat cultured cortical neurons, with an $IC_{50}$ value of $28.7{\pm}7.5\;{\mu}m$. Furthermore, the $Rg_3$ treatment not only significantly reduced DNA damage, but also dose-dependently attenuated 24-OH-chol-induced caspase-3 activity. To study the mechanisms underlying the in vitro neuroprotective effects of $Rg_3$ against 25-OH-chol-induced cytotoxicity, we also examined the effect of $Rg_3$ on intracellular $Ca^{2+}$ elevations in cultured neurons and found that $Rg_3$ treatment dose-dependently inhibited increases in intracellular $Ca^{2+}$, with an $IC_{50}$ value of $40.37{\pm}12.88\;{\mu}m$. Additionally, $Rg_3$ treatment dose-dependently inhibited apoptosis with an $IC_{50}$ of $47.3{\pm}14.2\;{\mu}m$. Finally, after confirming the protective effect of $Rg_3$ using a terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay, we found that $Rg_3$ is an active component in ginseng-mediated neuroprotection. These results collectively indicate that $Rg_3$-induced neuroprotection against 24-OH-chol in rat cortical neurons might be achieved via inhibition of a 24-OH-chol-mediated $Ca^{2+}$ channel. This is the first report to employ cortical neurons to study the neuroprotective effects of $Rg_3$ against 24-OH-chol. In conclusion, $Rg_3$ was effective for protecting cells against 24-OH-chol-induced cytotoxicity in rat cortical neurons. This protective ability makes $Rg_3$ a promising agent in pathologies implicating neurodegeneration such as apoptosis or neuronal cell death.

Changes of Prosapogenin Components in Tienchi Seng (Panax notoginseng) by Ultrasonic Thermal Fusion Process

이재범,양병욱,김도형,Dezhong Jin,고성권 한국생약학회 2021 Natural Product Sciences Vol.27 No.1

The purpose of this study is to develop a new method of producing tienchi seng (notoginseng, Panax notoginseng) extracts featuring high concentrations of the ginsenoside Rg3, Rg5, and Rg6, special components of Korean red ginseng. The chemical transformation from ginseng saponin glycosides to prosapogenin was analyzed by HPLC. Tienchi seng was heat-processed at 100oC and the optimum conditions were identified. The highest concentrations of total saponin (29.723%) and the ginsenoside Rg3 (1.769%), Rg5 (5.979%), and Rg6 (13.473%) were produced at 48 hours. Also, when tienchi seng was subjected to the ultrasonic thermal fusion (100oC) process, the concentrations of total saponin (30.578%), ginsenoside Rg3 (2.392%), Rg5 (6.614%), and Rg6 (13.017%) were highest at 36 hours. On the other hand, the 2-hour heat-processed extract and 2-hour ultrasonic thermal fusion-processed extract did not contain ginsenoside Rg3, Rg5, and Rg6. The ultrasonic thermal fusion process had an extraction yield that was approximately 1.26 times greater than that of the heat process. These results indicate that the highly functional tienchi seng extracts created through the ultrasonic thermal fusion process are more industrially useful than those produced using the heat process.