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標題: Black garlic aging process and its immunomodulatory functions
作者: Ting-Yi Lee
關鍵字: 熟成;抗過敏;抗發炎;黑蒜;褐變;高溫高濕;免疫調節;aging;anti-allergy;anti-inflammation;black garlic;browning;high temperature and high humidity;immunomodulation
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黑蒜為大蒜(Allium sativum L)利用高溫高濕自然發酵熟成,具有相對較高之活性成分,然目前對於黑蒜的熟成變化與免疫調節功能仍不甚清楚,因此本研究將生大蒜,透過不同的溫度、濕度與熟成天數等不同的條件控制(熟成條件:70℃, 80%相對溼度、70℃, 70%相對溼度、60℃, 80%相對溼度,並分別熟成0、10及20天),以製造出不同的黑蒜,探討熟成期間其內部成分變化,從中挑選出最佳熟成條件。結果顯示,綜合外觀觀察、水分含量變化及色澤變化比較,以70℃, 80% RH為最佳;褐變程度、游離還原糖與游離胺基酸結果比較,以70℃, 80%熟成20天變化最明顯;總多酚、類黃酮、皂苷含量變化顯示,70℃, 80%含量最高,綜合本研究結果,建議以70℃, 80%相對溼度熟成20天為黑蒜之最佳熟成條件。
進一步將最佳熟成黑蒜依序以正己烷、95%乙醇、熱水等低、中、高極性溶劑萃取,以生大蒜作為對照組,探討黑蒜不同萃取物對BALB/c小鼠初代脾臟細胞Th1/Th2細胞免疫調節之影響,以及在脂多醣(lipopolysaccharide, LPS)誘導發炎下對初代腹腔巨噬細胞之促/抗發炎細胞激素分泌之影響,並分析不同萃取物中的成分與免疫細胞受調節之細胞激素的相關性,以及對人類乳腺癌細胞生長之影響。結果顯示,黑蒜熱水萃取物(BGHWE)有效減少脾臟細胞IL-4細胞激素之分泌且有劑量反應關係。在LPS誘導巨噬細胞發炎模式下,發現生大蒜與熟成黑蒜乙醇萃取物皆能有效降低發炎程度,然兩者之間無顯著差異;黑蒜熱水萃取物可以顯著抑制IL-1β分泌以及顯著增加IL-10分泌,較生大蒜更具有抗發炎作用,整體評估黑蒜之免疫調節作用,以熱水萃取物效果最好,具有抗過敏與抗發炎的作用。相關性分析結果顯示,黑蒜熱水萃取物中之指標成分,並無何種單獨成分,對於黑蒜之免疫調節效果中具有特別明顯之貢獻,推測萃取物中各種未知成分之間,可能具有相互影響或互相干擾之作用,導致相關性減弱。另外,黑蒜熱水萃取物對MCF-7細胞生長無論直接與經由免疫細胞調節之間接作用皆無抑制MCF-7細胞生長之效果。

Black garlic is prepared from fresh raw garlic (Allium sativum L.) through aging at high temperature and high humidity for a period of time. It is presumed that black garlic has the better physiological function than that of raw garlic. However, changes in aging process and immunomodulatory effects of black garlic still remain unclear. To clarify the puzzle, raw garlic was treated at different controlled temperature and relative humidity (RH) for different incubation time (aging condition: 70℃ and 80% RH, 70℃ and 70% RH, as well as 60℃ and 80% RH for 0, 10 and 20 days, respectively) to produce different kinds of black garlic for investigating their changes in components during aging process. Based on the results, the best aging condition would be recommended. The results showed that 70℃ and 80% RH was the best aging condition according to the appearance, moisture content and color changes of aged black garlic. Aging at 70℃ and 80% RH for 20 days showed prominent changes in browning index, free reducing sugar content and free amino acid content. Aging at 70℃ and 80% RH had the highest content of total polyphenol, flavonoids and saponins in aged black garlic. Therefore, aging at 70℃ and 80% RH for 20 days was suggested to be the best aging condition for black garlic aging.
To evaluate immunomodulatory effects of aged black garlic, the best aged black garlic was sequentially extracted using n-hexane, 95% ethanol and hot water that were corresponding to low, medium and high polar solvents, respectively. Raw garlic was selected as a control in the experiment. Effects of different aged black garlic extracts on Th1/Th2 immune balance of splenocytes and pro-/anti-inflammatory cytokines secretion by lipopolysaccharide (LPS)-induced peritoneal macrophages form female BALB/c mice were investigated. The relationships between cytokines secretion by regulated immune cells and possible bioactive components in different aged black garlic extracts were analyzed. In addition, effects of different aged black garlic extract on human breast cancer MCF-7 cells were studied. The results showed that black garlic hot water extracts (BGHWE) had anti-allergic potential for it significantly reduced IL-4 secretion by splenocytes dose-dependently. Both ethanol extracts of aged black garlic and raw garlic reduced inflammatory responses in LPS-induced peritoneal macrophages, while the impact were similar to each other. BGHWE significantly suppressed IL-1β secretion but significantly enhanced IL-10 secretion, indicating that it had the strongest anti-inflammatory effects among different aged black garlic extracts. BGHWE had the better anti-inflammatory effect than that of raw garlic hot water extracts. BGHWE exhibited the best anti-allergic and anti-inflammatory potential among different extracts. However, there was no significant correlation between individual marker components in different extracts and cytokines secretion by regulated immune cells. It was assumed that unknown components exist in black garlic extracts might influence or interfere each other, resulting in a decrease of correlation. Unfortunately, BGHWE had no obvious effects on the growth of human breast cancer MCF-7 cells regardless of direct or indirect models.
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