JPH06211885A - New diterpene glycoside and tobacco smoking flavor improver containing the same as active ingredient - Google Patents

Abstract

PURPOSE:To obtain a low-volatile new compound excellent in long-term preservability, useful as e.g. a tobacco smoking flavor improver. CONSTITUTION:The objective compound of the formula {R1 and R2 are each sugar chain consisting of D-glycosyl or L-rhamnose; when R1 is o-beta-D-glycosyl, R2 is o-[alpha-L-rhamnopyranosyl(1 4)]-[alpha-L-rhamnopyranosyl(1 6)]-beta-D- glycopyranosyl; when R1 is o-alpha-L-rhamnopyranosyl(1 4)-#'-D-glycosyl, R2 is o-[beta-D-glycopyranosyl(1 2)]-[alpha-L-rhamnopyranosyl(1 6)]-beta-D-glycopyranosyl, etc.,}, which can be obtained by the following process: The leaves, etc., of a nicotiana plant belonging to Solanaceae are immersed in a polar solvent such as methanol, ethanol or water to carry out extraction, and the resultant extraction solvent concentrated and then put to porous resin column chromatography followed by high-performance liquid chromatography to perform separation of the objective compound.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel diterpene derivative and a flavor and taste improving agent for tobacco containing the derivative as an active ingredient.

[0002]

2. Description of the Related Art In recent years, cigarette smokers have tended to shift to low nicotine and low tar cigarettes (cigarettes). However, such a cigarette has a drawback in that the smoker does not have a sufficient satisfaction because the flavor and smoke amount are small when smoking.

In order to make up for such drawbacks, conventionally, many essential oil components have been isolated from leaf tobacco, and these have been added to cigarettes having a poor flavor and taste to enhance the flavor and smoke amount. ing.

However, since many essential oil components derived from leaf tobacco are easily volatilized, they are volatilized in a short period of time after perfuming a cigarette, and a sufficient flavor and smoke volume enhancing effect is obtained until smoking. Difficult to maintain.

The present invention has been made in view of the above points, and is a novel diterpene derivative capable of exhibiting a very excellent tobacco flavor and taste improving effect during smoking and being stable and maintaining the effect during storage. And a flavor and taste improving agent for tobacco, which comprises the derivative as an active ingredient.

[0006]

The present invention provides a diterpene glycoside represented by the following formula [1].

[0007]

[Chemical 3] (In the formula, R ₁ and R ₂ represent a sugar chain composed of D-glucose and L-rhamnose, and R ₁ is O-β-D.
-Glucosyl, R ₂ is O- [α-L-rhamnopyranosyl (1 → 4)]-[α-L-rhamnopyranosyl (1 → 6)]-β-D-glucopyranosyl, R ₁
Is O-α-L-rhamnopyranosyl (1 → 4) -β-D-
When it is glucosyl, R ₂ is O- [β-D-glucopyranosyl (1 → 2)]-[α-L-rhamnopyranosyl (1 → 6)]-β-D-glucopyranosyl, or O
-[Α-L-Rhamnopyranosyl (1 → 4)]-[α-L
-Rhamnopyranosyl (1 → 6)]-β-D-glucopyranosyl. The present invention also provides a flavor and taste improving agent for tobacco, which comprises the above-mentioned novel diterpene glycoside as an active ingredient.

The present invention will be described in more detail below.

The present inventors extensively searched for compounds effective in improving the flavor and taste of cigarettes, and as a result, it was found that cultivated tobacco (barley, native) and wild-type tobacco extracts were found. However, they have found an effective compound that is hard to volatilize but can give a flavor and taste to a cigarette upon burning.

The novel diterpene glycoside of the present invention (hereinafter,
This compound) is a leaf of Nicotiana plant of the Solanaceae family,
It is contained in flowers and stems, and in particular, tobacco plants such as Nicotiana tabacum (Valley species and native species), which is a cultivated species, and Nicotina umbratica, which is a wild species. Is contained in large amounts.

The present compound was obtained by extracting these Nicotiana plants by immersing them in a polar solvent such as methanol, ethanol or water and concentrating the resulting extract. The concentrate was subjected to a porous resin column chromatography, and then a high performance liquid chromatography (HPL).
It can be obtained by attaching to C) and separating.

When the present compound is used as a flavor and taste improving agent for tobacco, its addition amount is preferably in the range of, for example, 0.01 to 1000 ppm based on the cut weight of tobacco, but depending on the flavor and taste quality of the starting tobacco. Should be increased or decreased.

The flavor and taste improving agent for tobacco of the present invention comprises
In addition to the present compound, other compounds or natural products generally used for improving the flavor and taste of cigarettes may be contained.

[0014]

The present invention will be described in more detail with reference to the following examples.

Example 1 An example of the method for producing the diterpene glycoside of the present invention will be described.

First, 8 kg of fresh leaves of Burley seed tobacco were immersed in 36 liters (L) of methanol for 10 days. After filtering this extract, the filtrate was concentrated at 35 ° C. using a rotary evaporator. Then, the obtained concentrate was dissolved in 1 L of water, and this solution was washed twice with 1 L of hexane to remove the hexane-soluble portion.

The water-soluble portion thus obtained was subjected to column chromatography with a porous resin column to fractionate the eluate. That is, a column (diameter 80 mm, height 550 mm) was filled with 500 mL of DIAION HP20 (manufactured by Mitsubishi Kasei Co.), and water 10
L, a 20% ethanol aqueous solution (2.5 L) and ethanol (2.5 L) were sequentially eluted.

Of these, the ethanol elution fraction was concentrated, and a part of the obtained concentrate was subjected to HPLC under the operating conditions shown below to give glycoside I (101.7 mg) and glycoside. II (74.0 mg) was obtained.

Operating conditions of HPLC Column: YMC-Pack A-314 ODS (manufactured by Yamamura Chemical Co., Ltd.) Solvent; Methanol: Water = 8: 2 Flow rate; 1.0 ml / min Detector; RI Glycoside I in the above HPLC The retention times of glycoside II and glycoside II were 8.4 minutes and 10.9 minutes, respectively. Glycoside I
And glycoside II are normal-phase thin-layer plates (Mechle,
In thin layer chromatography (TLC) using Art 5642), anthron-sulfuric acid reagent developed a grayish black color, and each spot showed 1 spot. In this TLC, the Rf value of glycoside I was 0.26 and the Rf value of glycoside II was 0.34 (developing solvent; chloroform: methanol: water = 14: 6: 1).

In order to confirm the type of sugar in the sugar chain of glycoside I and glycoside II, glycoside I and glycoside II were hydrolyzed and then subjected to gas chromatography (GC). That is, 2 ml of 2N-trifluoroacetic acid was added to 1 mg of the test sample and hydrolyzed at 100 ° C for 1 hour. After the reaction solution was concentrated and dried, the concentrate was dissolved in 40 μl of an ethanethiol / trifluoroacetic acid (2: 1) solution and left at room temperature for 102 minutes to release sugar (free sugar) from the glycoside.
Was dithioacetalized. Then, 100 μl of viridine was added to the reaction solution to terminate the reaction, and trimethylsilyl chloride (BSTFA, trade name; PIERC) was added.
E CHEMICAL Co. , Made) and allowed to stand at 50 ° C. for 30 minutes to give a sugar as a dithioacetal-TMS derivative, and this derivative was subjected to GC according to the following operating conditions.
Analysis was carried out.

Operating conditions of GC column; DB-1 column temperature; 220 ° C. carrier gas; helium inlet temperature; 250 ° C. detection port temperature; 250 ° C. detector; FID. Dithioacetal-TM
When the retention time was compared with that of the S derivative, D-
Consistent with glucose and L-rhamnose. This revealed that glycoside I and glycoside II contained D-glucose and L-rhamnose. Furthermore, in order to confirm the structures of glycoside I and glycoside II,
¹³ C-NMR measurement (AM500, manufactured by Bruker)
Was carried out, and each carbon was assigned. The results of ¹³ C-NMR analysis of glycoside I and glycoside II were as follows.

¹³ C-NMR Carbon chemical shift (δpp of aglycone of glycoside I
m, in deuterated methanol) 115.86, 144.23, 81.43, 42.45, 23.46, 125.67, 13
5.82, 40.65, 27.50, 125.86, 135.28, 40.74, 27.0
8, 131.12, 132.29, 21.96, 23.22., 16.13, 16.23
, 68.11, chemical shift of carbon of sugar chain of glycoside I (δ ppm, in deuterated methanol); Glc; 99.25, 75.12, 76.78, 79.52, 76.34
, 62.04 Rham; 102.64, 72.26, 72.08, 73.63, 70.49.
, 17.83 Glc '; 100.90, 81.86, 77.94, 71.13, 76.45.
, 67.45 Rham '; 101.70, 72.01, 72.30, 73.89, 69.61
, 18.08 Glc ”; 104.65, 75.74, 77.58, 71.35, 78.04
, 62.67 Chemical shift of carbon of aglycone of glycoside II (δpp
m, in deuterated methanol) 115.78, 144.45, 81.84.,
42.65, 23.55, 125.63, 135.92, 40.69, 27.62, 12
5.90, 135.47, 40.78, 27.29, 131.25, 132.45, 21.6
7, 23.28, 16.11, 16.24, 67.71 Chemical shift of carbon of sugar chain of glycoside II (δ ppm, in deuterated methanol); Glc; 99.42, 75.31, 76.96, 79.75, 76.52
, 62.14 Rham; 102.86, 72.51, 72.22, 73.60, 70.65.
, 17.84 Glc '; 102.20, 75.52, 76.76, 79.30, 75.43
, 66.68 Rham '; 101.63, 72.22, 72.36, 74.02, 79.62.
, 18.17 Rham ”; 102.70, 72.45, 72.26, 73.79, 70.61.
From the above results, glycoside I and glycoside II have (3,6E, 10E, 14Z) -20-hydroxygeranyllinalool as an aglycone, and glycoside I has 2 mol of D.
-Glucose and 3 mol of L-rhamnose, glycoside II is 3
It was confirmed to have moles of D-glucose and 2 moles of L-rhamnose.

Furthermore, in order to confirm the molecular weights of glycoside I and glycoside II, FAB-MS measurement (KRATOS CONCEPT IIH, manufactured by Shimadzu Corporation, accelerating voltage; 8 kV, matrix; nitrobenzyl alcohol) was performed. It was The measurement results are shown below.

Glycoside I: 1107.5 (M + Na ⁺ ) Glycoside II; 1091.5 (M + Na ⁺ ) From the above analysis results, glycoside I and glycoside II are represented by the formula [1]. It was determined that R ₁ and R ₂ are the diterpene glycosides shown in Table 1.

[0025]

[Table 1] Example 2 Hereinafter, a second example of the method for producing the compound of the present invention will be described.

Nicotiana Ambratika's fresh leaves 8.8K
After g was immersed in 40 L of chloroform for about 10 seconds to remove the leaf surface resin, the residue was immersed in 50 L of methanol for 10 days. After filtering this extract, it was concentrated at 35 ° C. by a rotary evaporator. This concentrate is then added to water 1
It was dissolved in L and washed twice with 1 L of hexane to remove the hexane-soluble portion.

The water-soluble portion thus obtained was subjected to column chromatography using a porous resin column,
The eluate was fractionated. That is, 500 ml of DIAION HP20 (manufactured by Mitsubishi Kasei) was packed in a column (diameter 80 mm, height 550 mm) and eluted successively with 10 L of water, 1.5 L of 20% aqueous ethanol solution and 4 L of ethanol.

Of these, the ethanol-eluted fraction was concentrated, and a part of the obtained concentrate was subjected to HPLC under the operating conditions shown below to give glycoside III (67.8 mg). .

Operating conditions of HPLC Column: YMC-Pack A-314 ODS (manufactured by Yamamura Chemical Co., Ltd.) Solvent; Methanol; Water = 75: 25 Flow rate; 1.0 ml / min Detector; RI Here, glycoside III The retention time was 15.8 minutes. Further, glycoside III showed 1 spot by TLC using a normal phase thin metal plate (Art 5642, manufactured by Merck & Co., Inc.), which developed a grayish black color with the anthrone-sulfuric acid reagent. The Rf value of glycoside III was 0.34 (developing solvent; chloroform: methanol: water = 14: 10:
1).

In order to confirm the type of sugar in the sugar chain, glycoside
After hydrolyzing III, GC analysis was performed. That is,
2 ml of 2N-trifluoroacetic acid was added to 1 mg of the test sample, and 1
It was hydrolyzed at 00 ° C for 1 hour. After concentrating and drying the reaction mixture, ethanethiol / trifluoroacetic acid (2: 1)
The free sugar was dithioacetalized by dissolving it in 40 μl of the solution and leaving it for 10 minutes at room temperature. 100 μl of pyridine was added to the reaction solution to terminate the reaction, trimethylsilyl chloride (BSTFA) was added, and the temperature was 50 ° C.
The mixture was allowed to stand for 30 minutes at 30 ° C. to give a dithioacetal-TMS derivative of free sugar. This dithioacetal-T
The MS derivative was analyzed by GC under the following operating conditions.

Operating conditions of GC Kamura; DB-1 Kamura temperature; 220 ° C. carrier gas; helium inlet temperature; 250 ° C. detection port temperature; 250 ° C. detector; FID. Acetal-TM
When the retention time was compared with that of the S derivative, D-
Consistent with glucose and L-rhamnose. This revealed that glycoside III contained D-glucose and L-rhamnose. Furthermore, in order to confirm the structure of glycoside III, ¹³ C-NMR measurement (AM500, manufactured by Bruker) was performed to assign each carbon. The results of ¹³ C-NMR analysis of glycoside III were as follows.

¹³ C-NMR Chemical shift of carbon of aglycone of glycoside III (δp
pm in deuterated methanol) 115.60, 144.44, 81.45
, 42.63, 23.54, 125.80, 135.88, 40.74, 27.60
, 125.87, 135.42, 40.85, 27.24, 131.21, 132.4
1,21.66, 23.23, 16.11, 16.24, 67.71 Chemical shift of carbon in sugar chain of glycoside III (δppm,
In deuterated methanol); Glc; 99.50, 75.14, 78.22, 71.69, 77.53
, 62.81 Glc '; 102.17, 75.19, 76.70, 79.25, 75.39.
, 66.65 Rham '; 101.58, 72.17, 72.33, 73.97, 69.77.
, 18.15 Rham ”; 102.64, 72.45, 72.23, 73.76, 70.57.
From the above results, glycoside III has (3,6E, 10E, 14Z) -20-hydroxygeranyllinalool as an aglycone, and 2 mol of L-rhamnose with 2 mol of D-glucose. It was confirmed to have.

Further, in order to confirm the molecular weight of glycoside III, FAB-MS measurement (KRATOS manufactured by Shimadzu Corporation)
CONCEPT IIH type, accelerating voltage; 8 kV, matrix: 2-pyrrolidone). The measurement results are shown below.

Glycoside III: 945.45 (M + Na ⁺ ) From the above analysis results, glycoside III is a diterpene glycoside represented by the formula [1], and R ₁ and R ₂ are 1
It was decided to be shown in.

Example 3 Hereinafter, a third example of the method for producing the compound of the present invention will be described.

2.68 kg of fresh leaves of conventional seed tobacco were dipped in 4 L of chloroform for about 10 seconds to remove the leaf surface resin, and the residue was mixed with chloroform / methanol (1: 1) 4
It was immersed in L for 8 days and the extract was filtered. 0.9 parts of water was added to this extract, and chloroform: methanol: water = 1: 1.
The ratio was set to 1: 0.9, the lower layer was separated with a separating funnel, and the lower layer was concentrated with a rotary evaporator at 35 ° C.

The concentrate thus obtained was subjected to column chromatography using a porous resin column,
This eluate was fractionated. That is, the column (diameter 80 mm,
550 mm in height) was filled with 500 L of DIAION HP20 (manufactured by Mitsubishi Kasei), and 10 L of water was added to this column.
1.5 L of 20% aqueous ethanol solution and ethanol 1.
Elution was performed sequentially with 5 L.

Of these, after the ethanol elution fraction was concentrated, a column (diameter 3) packed with Sephadex LH20 (Pharmacia Fine Chemical Co.) was further packed.
Column chromatography (0 mm, height 600 mm) and the eluate was eluted with methanol 7
Fractionation into fractions was performed, and a portion of fraction 2 was subjected to HPLC under the operating conditions shown below to give glycoside IV (9.
3 mg) was obtained.

Operating conditions of HPLC Column: YMC-Pack A-314 ODS (manufactured by Yamamura Chemical Co., Ltd.) Solvent; Methanol: Water = 8: 2 Flow rate; 1.0 ml / min Detector; RI Here, glycoside IV The retention time was 14.0 minutes.
Glycoside IV is a thin layer of normal phase and (Merck, Art.
In TLC using 5642), anthron-sulfuric acid reagent developed a grayish black color, and one spot was shown. Glycoside
The Rf value of IV was 0.56 (developing solvent; chloroform: methanol: water = 14: 10: 1).

In order to confirm the type of sugar in the sugar chain, glycoside
After hydrolysis of IV, GC analysis was performed. That is, 2 ml of 2N-trifluoroacetic acid was added to 1 mg of the sample, and
Hydrolyzed at 0 ° C. for 1 hour. After concentrating and drying the reaction solution, ethanethiol / trifluoroacetic acid (2:
1) The solution was dissolved in 40 μl of the solution, and left at room temperature for 10 minutes to dithioacetalize the free sugar. After adding 100 μl of pyridine to the reaction solution to terminate the reaction,
Add trimethylsilyl chloride (BSTFA) and add 5
By leaving it at 0 ° C. for 30 minutes, as a dithioacetal-TMS derivative of free sugar, this derivative was subjected to GC analysis under the following operating conditions.

Operating conditions of GC column; DB-1 column temperature; 220 ° C. carrier gas; helium inlet temperature; 250 ° C. detection port temperature; 250 ° C. detector; FID. Dithioacetal-TM
When the retention time was compared with that of the S derivative, D-
Consistent with glucose and L-rhamnose. This revealed that glycoside IV contained D-glucose and L-rhamnose. Furthermore, in order to confirm the structure of glycoside IV, ¹³ C-NMR measurement (manufactured by Bruker, A
M500) was carried out to assign each carbon. The results of ¹³ C-NMR analysis of glycoside IV were as follows.

¹³ C-NMR Chemical shift of carbon of aglycone of glycoside IV (δpp
m, in heavy methanol) 115.78, 144.45, 81.84, 42.65, 23.55, 125.63, 135.9
2, 40.69, 27.62, 125.90, 135.47, 40.78, 27.29, 131.
25, 132.45, 21.67,23.28, 16.11, 16.24, 67.71 Chemical shift of carbon of sugar chain of glycoside IV (δ ppm, in heavy methanol); Glc; 99.42, 75.31, 76.96, 79.75, 76.52, 62.
14 Rham; 102.86, 72.51, 72.22, 73.60, 70.65, 1
7.84 Glc '; 102.20, 75.52, 76.76, 79.30, 75.43, 6
6.68 Rham '; 101.63, 72.22, 72.36, 74.02, 79.62, 1
8.17 Rham ”; 102.70, 72.45, 72.26, 73.79, 70.61, 1
7.84 From the above results, glycoside IV is aglycone (3,
6E, 10E, 14Z) -20-Hydroxygeranyl linalool and was confirmed to have 2 moles of D-glucose and 2 moles of L-rhamnose.

Further, in order to confirm the molecular weight of glycoside IV, FAB-MS measurement (manufactured by Shimadzu Corporation, KRATOS) was performed.
CONCEPT IIH type, accelerating voltage; 8 kV, matrix; nitrobenzyl alcohol). The measurement results are shown below.

Glycoside IV: 1091.5 (M + Na ⁺ ) From the above analysis results, glycoside IV is a diterpene glycoside represented by the formula [1], and R ₁ and R ₂ are shown in Table 1. It was decided to be shown in.

Example 4 The amounts of glycosides I, II, III and IV isolated in Examples 1, 2 and 3 were each 0.01 pp per unit weight of cut tobacco.
After appropriately dissolving in ethanol so as to obtain m and 1000 ppm, the mixture was uniformly added by spraying on the tobacco cuts of the cigarette leaf set. After this, room temperature (25
After being left to stand at (.degree. C.) and sufficiently adapted to cut tobacco, it was rolled up in the form of a cigarette to obtain perfume products I to IV with addition amounts of 0.01 ppm and 1000 ppm.

The obtained flavored products I to IV were subjected to a two-point discrimination test method with a flavorless product to which no compound was added. A sensory test was conducted. The results are shown in Table 2.

[0047]

[Table 2] * Numbers indicate the number of panel members who were considered better than unscented products.

As is clear from Table 2, in the fragrance products,
Both the added amount of 01 ppm and 1000 ppm increased both the flavor and the smoke amount, and it was confirmed that all of glycosides I to IV could exhibit the excellent effect of improving the tobacco flavor and taste.

[0049]

As described above, according to the novel diterpene glycoside of the present invention and the flavor enhancer for tobacco containing the glycoside as an active ingredient, a small amount is added to the cut tobacco. It is possible to impart or enhance the flavor and smoke amount to the cigarette, and since it has low volatility, the effect can be maintained until smoking. As a result, it is possible to easily improve the flavor quality of cigarettes aimed at low nicotine and low tar, and to provide a cigarette suitable for long-term storage.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Shizuo Suhara, Inventor Shizuo Suhara, 6-6 Umegaoka, Midori-ku, Yokohama, Kanagawa Japan Tobacco Inc. Tobacco Central Research Institute (72) Toshiaki Matsuzaki 6-2, Umegaoka, Midori-ku, Yokohama, Kanagawa Japan Tobacco Inc. Tobacco Central Research Institute

Claims (2)

[Claims] 1. A diterpene glycoside represented by the following formula [1]. [Chemical 1] (In the formula, R ₁ and R ₂ represent a sugar chain composed of D-glucose and L-rhamnose, and R ₁ is O-β-D.
-Glucosyl, R ₂ is O- [α-L-rhamnopyranosyl (1 → 4)]-[α-L-rhamnopyranosyl (1 → 6)]-β-D-glucopyranosyl, and R ₁
Is O-α-L-rhamnopyranosyl (1 → 4) -β-D-
When it is glucosyl, R ₂ is O- [β-D-glucopyranosyl (1 → 2)]-[α-L-rhamnopyranosyl (1 → 6)]-β-D-glucopyranosyl, or O
-[Α-L-Rhamnopyranosyl (1 → 4)]-[α-L
-Rhamnopyranosyl (1 → 6)]-β-D-glucopyranosyl. ) 2. A flavor and taste improving agent for tobacco, comprising a diterpene glycoside represented by the following formula [1] as an active ingredient. [Chemical 2] (In the formula, R ₁ and R ₂ represent a sugar chain composed of D-glucose and L-rhamnose, and R ₁ is O-β-D.
-Glucosyl, R ₂ is O- [α-L-rhamnopyranosyl (1 → 4)]-[α-L-rhamnopyranosyl (1 → 6)]-β-D-glucopyranosyl, R ₁
Is O-α-L-rhamnopyranosyl (1 → 4) -β-D-
When it is glucosyl, R ₂ is O- [β-D-glucopyranosyl (1 → 2)]-[α-L-rhamnopyranosyl (1 → 6)]-β-D-glucopyranosyl, or O
-[Α-L-Rhamnopyranosyl (1 → 4)]-[α-L
-Rhamnopyranosyl (1 → 6)]-β-D-glucopyranosyl. )